Methods for detecting Th1 cells

ABSTRACT

The inventors discovered that the adhesion molecule CAR, known to be localized in intracellular adhesion sites, functioned as an adhesion molecule for activated lymphocytes. Further, the inventors identified CARL, a novel CAR ligand expressed in lymphocytes, and clarified that the ligand was expressed selectively in Th1 cells. In addition, they found that anti-CAR antibodies could inhibit the adhesion of activated lymphocytes to CAR molecules. Thus, the present invention provides methods for detecting Th1 cells using CAR or anti-CARL antibodies, and methods of screening for inhibitors suppressing the adhesion of Th1 cells using the binding between CAR and CARL as an index. Furthermore, the present invention relates to methods of screening for inhibitors of the binding between CAR and CARL, antibodies that inhibit the binding between CAR and CARL, and therapeutic compositions comprising these antibodies. These are expected to be useful in diagnosing diseases, such as inflammation, in which infiltration of Th1 cells is involved, and in providing pharmaceutical agents for alleviating such diseases.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application is a divisional of U.S. application Ser. No.13/840,927, filed on Mar. 15, 2013, now U.S. Pat. No. 9,175,073, whichis a divisional of U.S. application Ser. No. 13/207,837, filed Aug. 11,2011, now abandoned, which is a divisional of U.S. application Ser. No.11/568,435, filed Mar. 28, 2008, which issued as U.S. Pat. No. 8,017,344on Sep. 13, 2011, which is a U.S. National Phase of PCT/JP2005/008150,filed Apr. 28, 2005, which claims the benefit of Japanese ApplicationNo. 2004-133093, filed on Apr. 28, 2004, the disclosures of which arehereby incorporated herein by reference in their entirety.

TECHNICAL FIELD

The present invention relates to methods for detecting Th1 cells, whichare helper T cells thought to be involved in cellular immunity.Specifically, the present invention relates to methods for detecting Th1cells using antibodies against coxsackie virus and adenovirus receptor(CAR) or against CAR ligand (CARL), a ligand on Th1 cells and binds toCAR. When the ratio between Th1 and Th2 cells in biological samples isexamined by the methods described above, patients from whom thebiological samples were collected can be diagnosed for atopic diseases.The present invention also relates to methods of screening forinhibitors of the binding between CAR and CARL. The present inventionfurther relates to antibodies that inhibit the binding between CAR andCARL, and cell adhesion inhibitors and therapeutic agents for contactdermatitis comprising these antibodies.

BACKGROUND ART

Immune reactions in the body take place locally, and are commonlycharacterized by the infiltration of immune cells. Specifically,efficient immune reactions are not achieved without adequatemobilization of the required immune cells to local sites. Further, thetypes of infiltrating immune cell vary depending on the tissues anddiseases. For example, a large number of Th1 cells producing interleukin(IL)-12, interferon (IFN)-γ, or such accumulate in joints withrheumatoid arthritis, while many Th2 cells producing IL-4, IL-5, or suchaccumulate in asthmatic lungs. By elucidating the cell infiltrationmechanism allowing such selective cell infiltration, it is thought thatinfiltration of specific cells can be suppressed and the diseases ofimmune reactions can thereby be controlled.

The infiltration of immune cells is roughly divided into four steps: (1)loose contact with vascular endothelial cells via secretin; (2) integrinactivation by chemokine receptors; (3) strong adhesion to vascularendothelial cells via integrins; and (4) extravascular migration throughintercellular spaces between vascular endothelial cells. The molecularmechanisms underlying the first three steps have been elucidated;however, the extravascular migration is still poorly understood.

JAM-A and PECAM-1 have recently been reported as molecules involved inextravascular migration. JAM-A and PECAM-1 are cell membrane proteinsbelonging to the immunoglobulin superfamily (IgSF), and function as celladhesion molecules.

The JAM family, comprising JAM-A, JAM-B, and JAM-C, comprise twoextracellular Ig-like domains and are reported as being expressed inepithelial cells, endothelial cells, leukocytes, platelets, and thelike. In addition to homophilic binding, binding to integrin αLβ2 hasbeen reported for JAM-A. Binding to integrin α4β1 and JAM-C, in additionto homophilic binding, have been reported for JAM-B. JAM-C has beenreported as binding to JAM-B and integrin αMβ2, but not as binding in ahomophilic manner. Thus, through such homophilic or heterophilicbinding, JAM family molecules are involved in adhesion betweenendothelial cells, between leukocytes and endothelial cells, and betweenplatelets and endothelial cells.

In addition to the JAM family, many other molecules comprising twoextracellular Ig-like domains exist. There are reports that some ofthese show similar localizations to molecules of the JAM family. Forexample, the coxsackie and adenovirus receptor (CAR) was identified as areceptor for coxsackie viruses and adenoviruses and is localized intight junctions and adherence junctions in epithelial cells andendothelial cells (Non-Patent Documents 1 and 2). Further, theendothelial-selective adhesion molecule (ESAM) was identified as amolecule selectively expressed only in endothelial cells, but was thenreported as being expressed in platelets as well.

To control diseases involving immune cells, it is extremely important toinvestigate whether such molecules belonging to IgSF are associated withthe adhesion of epithelial cells, endothelial cells, leukocytes, andplatelets, and to elucidate the mechanism underlying cell infiltration.

[Non-Patent Document 1] Cohen C. J., Shieh J. T., Pickles R. J., OkegawaT., Hsieh J. T., Bergelson J. M., Proc. Natl. Acad. Sci. USA, (2001)98(26):15191-15196.

[Non-Patent Document 2] Walters R. W., Freimuth P., Moninger T. O.,Ganske I., Zabner J., Welsh M. J., Cell, (2002) 110(6):789-799.

[Non-Patent Document 3] Moog-Lutz C., Cave-Riant F., Guibal F. C., BreauM. A., Di Gioia Y., Couraud P. O., Cayre Y. E., Bourdoulous S., Lutz P.G., Blood, (2003) 102(9):3371-3378.

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

Cell adhesion molecules with the ability to adhere to specificlymphocytes are thought to contribute to the selective cellularinfiltration of those lymphocytes, and thus by identifying these celladhesion molecules and their ligands, it is expected that cellularinfiltration of specific lymphocytes can be suppressed and the diseasesof immune reactions can be controlled. A purpose of the presentinvention is thus to search for cell adhesion molecules with the abilityto mediate cell adhesion to specific lymphocytes, to identify ligandsfor these molecules, and to discover uses for these adhesion molecules,ligands, and antibodies against them.

To search for cell adhesion molecules with the ability to adhere toactivated lymphocytes, the present inventors conducted cell adhesionexperiments on activated lymphocytes using chimeric molecules formedbetween secretory alkaline phosphatases and the extracellular domains ofcandidate molecules belonging to IgSF. As a result, CAR was revealed tofunction as an adhesion molecule for activated lymphocytes.

Next, monoclonal antibodies against CAR were produced, and three typesof CAR-specific antibodies (4C9, 5E9, and 5G11) were obtained. Celladhesion mediated by homophilic binding of CAR was inhibited by 5G11 andincreased by 4C9 and 5E9. Moreover, cell adhesion between CAR andactivated lymphocytes was inhibited by 4C9 and 5G11, but suppression wasnot observed with 5E9. It was thus suggested that the activity ofantibodies in inhibiting cell adhesion depends on the antibody bindingsite.

CAR expression in activated lymphocytes was investigated using theanti-CAR antibodies. Expression of CAR protein was undetectable on cellsurfaces, and expression of CAR mRNAs was also not detectable. Thissuggested that the homophilic binding of CAR did not mediate the celladhesion of activated lymphocytes, and that an unknown ligand(s) for CARis expressed in activated lymphocytes. Thus, first, the possibility thatthe unknown ligand for CAR was an integrin was examined. The effect oncell adhesion of antibodies against CD11a, CD18, CD29, CD49d, CD51, andCD61 was assessed. However, none of the antibodies inhibited adhesionbetween CAR and activated lymphocytes.

Moreover, to identify the unknown CAR ligands, the state of lymphocytesthat adhere to CAR was investigated. Activated lymphocytes were revealedto adhere to CAR when stimulated with IL-2, but not when stimulated withIL-15. Neither cells of the T cell line TK1, nor resting CD4+T cellsadhered to CAR. Furthermore, binding to the cell surface of variouslymphocytes was examined using chimeric proteins (CAR-AP) formed betweenalkaline phosphatase (AP) and extracellular regions of CAR. CAR-AP boundto IL-2-stimulated activated lymphocytes, but not to IL-15-stimulatedactivated lymphocytes, nor to cells of the T cell line TK1, nor toresting CD4+ T cells.

Next, the possibility that unknown CAR ligands were IgSF members wasinvestigated. Mouse Ensembl database was searched to investigate thechromosomal positions of CAR and the molecules of the JAM family. Thesemolecules formed discrete clusters on chromosomes 1, 9, and 16. 50 IgSFmolecules existing near CAR and the molecules of the JAM family wereselected, and their expression in various lymphocytes was investigatedusing real-time PCR. The results showed that ENSMUSG0000048534 exhibitedan expression pattern that correlated to the adhesion activity to CAR.Then, to examine whether or not ENSMUSG0000048534 was an unknown CARligand, chimeric proteins formed between the extracellular domain ofthis molecule and alkaline phosphatase were prepared and examined forthe activity of adhering to B300 cells and CAR-expressing B300 cells. Asa result, only CAR-expressing B300 cells adhered, and adhesion wasinhibited by 5G11. These results revealed that ENSMUSG0000048534 was anovel CAR ligand on lymphocytes, and ENSMUSG0000048534 was thus namedCARL (CAR ligand).

A gene encoding CARL was found on mouse chromosome 9 (46.9 Mb). The geneencoding CARL and a mouse cDNA sequence of unknown function deposited inGenBank as “similar to AMICA (BC050133)” matched. A gene for a humanhomolog of CARL is thought to be the human cDNA sequence AMICA(AY138965) deposited in GenBank, which is located on the mousechromosome homology region on human chromosome 11 (117.6 Mb) and has ahigh amino acid sequence homology, but whose function is unknown. Ahuman AMICA molecule lacking a portion of the amino acid sequence (humanJAML (A7515553)) has been recently reported as an adhesion moleculeexpressed in bone marrow-derived cells (Non-Patent Document 3). Analysesof protein-protein interactions using BIAcore revealed that CAR and CARLextracellular domains directly bound and their association affinityconstant was 4.8 nM. In addition, it was shown that CARL was selectivelyexpressed in Th1 cells among lymphocytes, and that CAR selectivelyadhered to Th1 cells among lymphocytes in a manner dependent on CARLexpression.

Further, cells expressing CARL lacking one of the two putative Ig-likedomains (domains 1 and 2) were each prepared and tested for theiractivity in adhering to the CAR-AP chimeric proteins. As a result, B300cells expressing full-length CARL and B300 cells expressing CARL lackingdomain 2 adhered to CAR-AP chimeric proteins, while B300 cellsexpressing CARL lacking domain 1 did not. Thus, domain 1 of CARL wasrevealed to be required for binding between CAR and CARL.

Next, #3 monoclonal antibody specific to CARL was obtained. CARLexpression in Th1 and Th2 cells was investigated using this #3 antibody,revealing a strong expression selectively in Th1 cells. The #3 antibodyinhibited the adhesion of CAR-expressing B300 cells to CARL-AP proteins.Further, the #3 antibody bound to CARL on the membrane of B300 cellsexpressing full-length CARL and B300 cells expressing CARL lackingdomain 2, but did not bind to those of B300 cells expressing CARLlacking domain 1. Thus the #3 antibody, which has the activity ofinhibiting binding was revealed to recognize domain 1, which is requiredfor binding between CAR and CARL.

CARL expression in cells other than Th1 cells was investigated, and CARLwas found to be strongly expressed in neutrophils. Further, when theproduced #3 anti-CARL antibody was used in a mouse model for contactdermatitis to conduct therapeutic experiments, a therapeutic effect wasobserved.

Human CARL-AP chimeric proteins were tested for their adhesion activityto B300 cells expressing human CAR, and binding between human CAR andhuman CARL was detected. Likewise, human CAR-AP chimeric proteins andhuman CARL-AP chimeric proteins were tested for adhesion activity toB300 cells expressing human CARL, and binding between human CAR andhuman CARL was detected. Thus, the phenomena observed in mice were alsoconfirmed in humans. Further, monoclonal antibodies against human CARLwere prepared, and antibodies that inhibited the binding of CARL toCAR-expressing cells were discovered. As was the case in mice,anti-human CARL antibodies were also shown to have an activity ofinhibiting cell adhesion and a therapeutic activity against contactdermatitis.

To summarize the above, the present inventors showed that: CAR functionsas an adhesion molecule for lymphocytes; CARL is a novel CAR ligand onlymphocytes; CARL is expressed selectively in Th1 cells; Th1 cellsselectively adhere to CAR; domain 1 is required for binding between CARand CARL; and human and mouse CAR and CARL show similar characteristics.Thus, the present invention relates to methods and kits for detectingTh1 cells, which use the direct interaction between CAR and CARL. Inanother embodiment, the present invention provides methods for detectingTh1 cells by using antibodies against CARL, which was identified as anovel ligand on Th1 cells. By such detection of Th1 cells, the ratiobetween Th1 and Th2 cells in biological samples can be examined, andsubjects from whom the biological samples were collected can bediagnosed for atopic diseases. The present invention thus relates tomethods for examining the ratio between Th1 and Th2 cells, and methodsfor diagnosing atopic diseases based on the examined ratio.

In yet another embodiment, the present invention relates to methods ofscreening for inhibitors of the binding between CAR and CARL. Thepresent invention also relates to antibodies that inhibit the bindingbetween CAR and CARL, and to compositions that inhibit cell adhesion,which comprise such antibodies, for example, compositions for treatingcontact dermatitis.

More specifically, the present invention provides the following:

-   [1] a method for detecting a Th1 cell, which comprises the steps of:    -   (a) contacting a cell sample comprising a lymphocyte with a CAR;        and    -   (b) detecting a cell that bound to a CAR in step (a);        wherein a CAR is a protein that binds to a polypeptide        comprising the amino acid sequence of SEQ ID NO: 1 or 2, and is        a protein comprising the amino acid sequence of any one of:    -   (1) an amino acid sequence of a natural CAR;    -   (2) an amino acid sequence encoded by a polynucleotide that        hybridizes under stringent conditions to a polynucleotide        encoding a natural CAR;    -   (3) an amino acid sequence with a deletion, substitution,        addition, or insertion of one or more amino acid residues in the        amino acid sequence of a natural CAR;    -   (4) an amino acid sequence with 90% or more homology to an amino        acid sequence of a natural CAR;    -   (5) an amino acid sequence comprising an extracellular domain of        an amino acid sequence of an above (1) to (4); or    -   (6) a fusion amino acid sequence of a marker protein and a        protein of an above (1) to (5);-   [2] the method of [1], wherein the CAR is bound to a carrier;-   [3] a kit for detecting a Th1 cell, which comprises a CAR as a    detection reagent;-   [4] the kit of [3], wherein the CAR is bound to a carrier;-   [5] a method for detecting a Th1 cell, which comprises the steps of:    -   (a) contacting a cell sample comprising a lymphocyte with an        anti-CARL antibody; and    -   (b) detecting a cell that bound to the anti-CARL antibody in        step (a);        wherein the anti-CARL antibody is an antibody that binds        specifically to a CARL, and wherein the CARL is a protein that        binds to a natural CAR and is the protein of any one of:    -   (1) a protein comprising the amino acid sequence of SEQ ID NO:        1;    -   (2) a protein comprising an extracellular domain of the amino        acid sequence of SEQ ID NO: 1;    -   (3) a protein comprising the amino acid sequence of SEQ ID NO:        2;    -   (4) a protein comprising an extracellular domain of the amino        acid sequence of SEQ ID NO: 2;    -   (5) a protein comprising an Ig-like domain 1 of an amino acid        sequence of an above (1) to (4);    -   (6) a protein encoded by a polynucleotide that hybridizes under        stringent conditions to the cDNA sequence of SEQ ID NO: 3 or 4;    -   (7) a protein comprising an amino acid sequence encoded by a        polynucleotide that hybridizes under stringent conditions to a        polynucleotide encoding an amino acid sequence of a protein of        the above (1) to (5);    -   (8) a protein comprising an amino acid sequence with 90% or more        homology to an amino acid sequence of a protein of the above (1)        to (5); or    -   (9) a protein comprising an amino acid sequence with a deletion,        substitution, addition, or insertion of one or more amino acid        residues in an amino acid sequence of a protein of the above (1)        to (5);-   [6] the method of [5], wherein the anti-CARL antibody is bound to a    carrier;-   [7] a method for examining the ratio between Th1 cells and Th2    cells, which comprises the method of [1] or [5];-   [8] a method for determining whether a subject from whom a cell    sample is collected is affected with an atopic disease based on the    ratio between Th1 cells and Th2 cells, which is examined by the    method of [7];-   [9] a kit for detecting a Th1 cell, which comprises an anti-CARL    antibody as a detection reagent;-   [10] the kit of [9], wherein the anti-CARL antibody is bound to a    carrier;-   [11] the kit of [9] that determines whether a subject from whom a    cell sample is collected is affected with an atopic disease;-   [12] a method of screening for an inhibitor of the binding between a    CAR and a CARL, which comprises the steps of:    -   (a) contacting a CAR and a CARL in the presence of a test        substance;    -   (b) detecting the binding between the CAR and the CARL in step        (a);    -   (c) comparing the degree of binding between the CAR and the        CARL, detected in step (b), with that in the absence of the test        substance; and    -   (d) selecting as an inhibitor to the binding between CAR and        CARL a test substance that suppresses the binding between the        CAR and the CARL compared to in the absence of the test        substance;        wherein the CARL is a protein that binds to a natural CAR, and        is the protein of any one of:    -   (1) a protein comprising the amino acid sequence of SEQ ID NO:        1;    -   (2) a protein comprising an extracellular domain of the amino        acid sequence of SEQ ID NO: 1;    -   (3) a protein comprising the amino acid sequence of SEQ ID NO:        2;    -   (4) a protein comprising an extracellular domain of the amino        acid sequence of SEQ ID NO: 2;    -   (5) a protein comprising an Ig-like domain 1 of the amino acid        sequence of an above (1) to (4);    -   (6) a protein encoded by a polynucleotide that hybridizes under        stringent conditions to the cDNA sequence of SEQ ID NO: 3 or 4;    -   (7) a protein comprising an amino acid sequence encoded by a        polynucleotide that hybridizes under stringent conditions to a        polynucleotide encoding an amino acid sequence of a protein of        the above (1) to (5);    -   (8) a protein comprising an amino acid sequence with a deletion,        substitution, addition, or insertion of one or more amino acid        residues in the amino acid sequence of a protein of an above (1)        to (5);    -   (9) a protein comprising an amino acid sequence with 90% or more        homology to an amino acid sequence of a protein of an above (1)        to (5); or    -   (10) a fusion protein between a marker protein and a protein of        the above (1) to (8); and wherein the CAR is a protein that        binds to a polypeptide comprising the amino acid sequence of SEQ        ID NO: 1 or 2, and is a protein comprising an amino acid        sequence of any one of:    -   (11) an amino acid sequence of a natural CAR;    -   (12) an amino acid sequence encoded by a polynucleotide that        hybridizes under stringent conditions to a polynucleotide        encoding a natural CAR;    -   (13) an amino acid sequence with a deletion, substitution,        addition, or insertion of one or more amino acid residues in the        amino acid sequence of a natural CAR;    -   (14) an amino acid sequence comprising an extracellular domain        of an amino acid sequence of an above (11) to (13); or    -   (15) a fusion amino acid sequence of a marker protein and the        protein of the above (14);-   [13] the method of [12], wherein either the CAR or the CARL is bound    to a carrier;-   [14] the method of [12], wherein the CAR and/or the CARL are    expressed in a host cell using an expression vector;-   [15] an antibody that inhibits the binding between a CAR and a CARL,    wherein a CARL is a protein of any one of:    -   (1) a protein comprising the amino acid sequence of SEQ ID NO:        1;    -   (2) a protein comprising an extracellular domain of the amino        acid sequence of SEQ ID NO: 1;    -   (3) a protein comprising the amino acid sequence of SEQ ID NO:        2;    -   (4) a protein comprising an extracellular domain of the amino        acid sequence of SEQ ID NO: 2;    -   (5) a protein comprising an Ig-like domain 1 of an amino acid        sequence of an above (1) to (4);    -   (6) a protein encoded by a polynucleotide that hybridizes under        stringent conditions to the cDNA sequence of SEQ ID NO: 3 or 4;    -   (7) a protein comprising an amino acid sequence encoded by a        polynucleotide that hybridizes under stringent conditions to a        polynucleotide encoding an amino acid sequence of a protein of        an above (1) to (5);    -   (8) a protein comprising an amino acid sequence with 90% or more        homology to an amino acid sequence of a protein of an above (1)        to (5); or    -   (9) a protein comprising an amino acid sequence with a deletion,        substitution, addition, or insertion of one or more amino acid        residues in the amino acid sequence of a protein of an above (1)        to (5);-   [16] the antibody of [15], wherein the CAR and the CARL are derived    from a human;-   [17] the antibody of [15] or [16], wherein the antibody is an    anti-CAR antibody;-   [18] the antibody of [17], wherein the anti-CAR antibody is produced    by hybridoma @mCAR:5E9-1-1 deposited under Accession Number: FERM    BP-10317, hybridoma @mCAR:5G11-1-1-11 deposited under Accession    Number: BERM BP-10318, or hybridoma @mCAR:4C9-1-1 deposited under    Accession Number: FERM BP-10320;-   [19] the antibody of [15] or [16], wherein the antibody is an    anti-CARL antibody;-   [20] the antibody of [19], wherein the anti-CARL antibody is    produced by hybridoma @mCARL:#3.11 deposited under Accession Number:    FERM BP-10319;-   [21] a cell adhesion inhibitor comprising the antibody of any one of    [15] to [20]; and-   [22] a therapeutic agent for contact dermatitis, which comprises the    antibody of any one of [15] to [20].

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing that CAR functions as an adhesion moleculefor activated T cells.

FIG. 2 shows the effect of anti-CAR antibodies on cell adhesion mediatedby homophilic binding of CAR.

FIG. 3 is a diagram and a photograph showing that CAR is not expressedon activated lymphocytes.

FIG. 4 shows that the activity of cell adhesion to CAR varies dependingon the condition of lymphocyte stimulation.

FIG. 5 shows the expression pattern of CAR ligand (CARL).

FIG. 6 shows cell adhesion between CAR and CAR ligand (CARL), and theinhibition of cell adhesion by anti-CAR antibodies.

FIG. 7 is a diagram showing the amino acid sequence of CARL (SEQ IDNO:1).

FIG. 8 is a graph showing the Kd value between CAR and CARL.

FIG. 9 shows that CAR selectively adheres to Th1 cells via binding withCARL, which is expressed selectively in Th1 cells.

FIG. 10A is a graph showing the inhibition by anti-CAR antibodies of thecell adhesion of Th1 cells to CAR.

FIG. 10B shows the expression of CARL in cells other than CD4-positive Tcells.

FIG. 11 shows that the region of CARL required for binding to CAR is thefirst Ig-like domain of CARL.

FIG. 12 shows the inhibition of cell adhesion by anti-CARL antibodies.

FIG. 13 shows that human CAR and human CARL bind to each other.

FIG. 14 shows the inhibition of cell adhesion by antibodies againsthuman CARL.

FIG. 15 is a graph showing the therapeutic effects of anti-CARL antibodyon contact dermatitis.

BEST MODE FOR CARRYING OUT THE INVENTION

Methods for Detecting Th1 Cells Using CAR

The present invention demonstrated that CAR binds to Th1 cells viadirect binding with CARL, which is specifically expressed on Th1 cells.The present invention thus relates to methods for detecting Th1 cellsusing CAR. Specifically, the present invention provides methods fordetecting Th1 cells, which comprise the steps of:

-   (a) contacting cell samples comprising lymphocytes with CAR; and-   (b) detecting the cells that bound to CAR in step (a).

The “cell samples comprising lymphocytes” in the present detectionmethods are not particularly limited, and examples include blood cellsamples expected to comprise lymphocytes, such as bone marrow andperipheral blood. Peripheral blood is particularly preferred forconvenience of collection.

Cell samples are typically contacted with CAR by adding polypeptides toreaction solutions comprising cells. For example, the cell adhesionbuffer (RPMI1640, 0.5% BSA, and 20 mM HEPES (pH 7.4)) described inExample 4 can be used as a reaction solution comprising cells. However,the present invention is not limited to this, and contact may also becarried out in any reaction solutions, as long the stability of CAR andTh1 cells is maintained and their binding is not inhibited.

Herein, “CAR” and “CAR polypeptide” comprise human proteins comprisingamino acid sequences (NP_001329; SEQ ID NO:17) encoded by the nucleotidesequence deposited in GenBank under accession number NM_001338 as wellas their allelic variants, splicing variants, and natural CARs, such asproteins derived from mammals other than humans and which correspond tothese proteins. Further, in addition to the natural CARs describedabove, “CAR” and “CAR polypeptide” comprise non-natural polypeptidesfunctionally equivalent to these CARs. “CAR” and “CAR polypeptide”comprise, for example, proteins with an altered amino acid sequence fromnatural CARs (altered CAR) and CARs produced as recombinant proteins(recombinant CARs). More specifically, “CAR” and “CAR polypeptide”comprise:

-   (1) amino acid sequences of natural CARs;-   (2) amino acid sequences encoded by polynucleotides that hybridize    under stringent conditions with polynucleotides encoding natural    CARs;-   (3) amino acid sequences with a deletion, substitution, addition, or    insertion of one or more amino acid residues in the amino acid    sequence of a natural CAR;-   (4) amino acid sequences with 90% or higher homology to an amino    acid sequence of a natural CAR;-   (5) amino acid sequences comprising an extracellular domain of an    amino acid sequence of the above (1) to (4); and-   (6) amino acid sequences obtained by fusing a marker protein with a    protein described in the above (1) to (5).

In the present description, “CAR” and “CAR polypeptide” are usedinterchangeably.

Mammals other than humans from which natural CARs are derived comprise,for example, mice (GenBank NP_034118; SEQ ID NO:18), rats, rabbits,dogs, horses, cats, pigs, bovine, goats, sheep, and primates such asmonkeys, gorillas, and chimpanzees. Allelic variants and the proteinsderived from these mammals can be obtained, for example, by obtainingcDNAs or genes encoding the allelic variants or proteins from cDNAlibraries, genomic libraries, or such using probes or primers producedbased on the nucleotide sequences or amino acid sequences of CARdescribed above and applying known techniques such as hybridization andPCR (Sambrook et al. (1989) Molecular Cloning: A laboratory manual, 2nded., Vol. 1-3, Cold Spring Harbor Laboratory Press; Current Protocols inMolecular Biology, John Wiley & Sons (1987-1997); DNA Cloning 1: coretechniques, a practical approach 2nd ed., Oxford Univ. (1995)), thenexpressing these genes. Alternatively, CAR may also be prepared byconventional protein synthesis methods based on known amino acidsequences.

As described above, “CAR” and “CAR polypeptide” comprise the naturalCARs described above as well as polypeptides functionally equivalent tothese CARs. The functions of CARs can be confirmed, for example, basedon binding with CARLs of the animals, such as the protein comprising ofthe amino acid sequence of SEQ ID NO: 1 in case of mice or SEQ ID NO: 2in case of humans. Proteins sharing biological activity are known togenerally be conserved evolutionarily at the amino acid sequence level,as well as at the level of the genes encoding the proteins. Thus,examples of polypeptides functionally equivalent to natural CARscomprise proteins exhibiting high amino acid sequence homology to anatural CAR, particularly in their active sites. Human AMICA, acounterpart of mouse CARL that binds to CAR, had a homology of 37.4%over the entire amino acid sequence. In the present invention, highamino acid sequence homology means, for example, an identity of 30%,35%, 40%, or 50% or more, preferably 60% or more, more preferably 70% ormore (for example, 80%, 90%, or 95% or more). Herein, the term “%identity” is defined as the percentage of matching between amino acidresidues (conservative substitutions not comprised in “matching”) whentwo amino acid sequences are aligned, permitting spaces as required toyield a maximal value between the two sequences. Such amino acidsequence % identity can be calculated using known software (BLAST (seeAltschul et al. (1990) J. Mol. Biol. 215:403-410; on the world wide webat ncbi.nlm.nih.gov.), BLAST-2, MegAlign, and such). One skilled in theart can appropriately determine each adjustable parameter required forthe calculation of identity using such known software, after consideringthe sensitivity or the like.

Genes encoding proteins that exhibit high amino acid sequence homologyand comparable activity to a natural CAR can be obtained usingconventional techniques, such as hybridization and PCR, using probes orprimers produced based on known amino acid sequences or gene sequencesof a natural CAR (Sambrook et al. (1989) Molecular Cloning: A laboratorymanual 2nd ed., Vol. 1-3, Cold Spring Harbor Laboratory Press). Proteinswith biological activities equivalent to those of a natural CAR can beobtained by expressing these genes. Thus, the “CAR” and “CARpolypeptide” described in the present description comprise proteinscomprising amino acid sequences encoded by polynucleotides thathybridize under stringent conditions to polynucleotides encoding anatural CAR. Examples of hybridization conditions for use in the presentinvention include “2×SSC, 0.1% SDS, 50° C.”, “2×SSC, 0.1% SDS, 42° C.”,and “1×SSC, 0.1% SDS, 37° C.”. Conditions of higher stringency include“2×SSC, 0.1% SDS, 65° C.”, “0.5×SSC, 0.1% SDS, 42° C.”, and “0.2×SSC,0.1% SDS, 65° C.”. More specifically, a method that uses the Rapid-hybbuffer (Amersham Life Science) can be carried out by performingpre-hybridization at 68° C. for 30 minutes or more, adding a probe toallow hybrid formation at 68° C. for one hour or more, washing threetimes in 2×SSC/0.1% SDS at room temperature for 20 minutes per wash,washing three times in 1×SSC/0.1% SDS at 37° C. for 20 minutes per wash,and finally washing twice in 1×SSC/0.1% SDS at 50° C. for 20 minutes perwash. This may also be carried out using, for example, the ExpresshybHybridization Solution (CLONTECH), by performing pre-hybridization at55° C. for 30 minutes or more, then adding a labeled probe andincubating at 37° C. to 55° C. for one hour or more, washing three timesin 2×SSC/0.1% SDS at room temperature for 20 minutes per wash, andwashing once at 37° C. for 20 minutes with 1×SSC/0.1% SDS. Herein,conditions of higher stringency can be achieved by setting a hightemperature (for example, 60° C. or 68° C.) for pre-hybridization,hybridization, and the second wash. In addition to salt concentration ofthe buffer and temperature, one skilled in the art can also integrateother hybridization factors, such as probe concentration, probe length,nucleotide sequence composition of the probe, and reaction time, toobtain CAR isoforms and allelic variants, and corresponding genesderived from other species. Molecular Cloning: A Laboratory Manual2^(nd) ed. (Cold Spring Harbor Press (1989)), Current Protocols inMolecular Biology (John Wiley & Sons (1987-1997)), DNA Cloning 1: CoreTechniques, A Practical Approach 2^(nd) ed. (Oxford University (1995))and such can be used as laboratory manual for the hybridization method.

In the present description, “CAR” and “CAR polypeptide” further compriseproteins with altered amino acid sequences from a natural CAR describedabove. Examples of such an “altered CAR” and “altered CAR polypeptide”include proteins in which a region that is not involved in the bindingof a natural CAR with CARL has been deleted. Examples of particularlypreferred proteins in which a portion of a natural CAR has been deletedinclude polypeptides comprising an amino acid sequence comprising anextracellular domain of a natural CAR. Protein domains can be predictedat domain searching sites, for example, at on the world wide web atsmart.embl-heidelberg.de/. Other than the above, the altered CARs andaltered CAR polypeptides of the present invention also comprise proteinsin which one or more amino acid residues of a natural CAR or a suitablepolypeptide chain is attached to a natural CAR, and proteins comprisingan amino acid sequence with a substitution or insertion of one or moreamino acid residues in the amino acid sequence of the natural CAR.Methods for deleting, adding, substituting, or inserting into a proteinwith a known amino acid sequence, arbitrary amino acid residues of thesequence are known. Such proteins can be produced, for example, byperforming a site-directed mutagenesis, which is a known technique (see,for example, Nucleic Acid Research, Vol. 10, No. 20, p. 6487-6500,1982), to DNAs encoding them. In the present description, the phrase“one or more amino acids” means a number of amino acids that can beadded, deleted, or substituted by performing site-directed mutagenesisone or more times. Site-directed mutagenesis can be performed, forexample, as described below using synthetic oligonucleotide primerscontaining the desired mutations in the complementary strand to thephage DNAs to be mutagenized. Specifically, the aforementioned syntheticoligonucleotides are used as primers to synthesize strands complementaryto the phage DNAs, and host cells are transformed with the obtaineddouble-stranded DNAs. The cultures of transformed bacteria are plated onagar, and single cells containing the phages are allowed to formplaques. In such cases, theoretically 50% of the new colonies containphages comprising the mutations in the single-stranded phage DNAs, whilethe remaining 50% comprise the original sequence. The obtained plaquesare hybridized with synthetic probes labeled by kinase treatment at atemperature at which those that completely match with the DNAscomprising the above desired mutations would hybridize, but thosecomprising the original strand would not hybridize. Then, those plaquesthat hybridize to the probe are selected, and DNAs are recovered afterculture.

In addition to the above-described site-directed mutagenesis, methodsfor substituting, deleting, or inserting one or more amino acids intothe amino acid sequences of biologically active peptides, such asenzymes, without loss of their activities, include methods in whichgenes are treated with mutagens and methods in which genes areselectively cleaved, selected nucleotides are deleted, added, orsubstituted, and the genes are ligated.

The CARs and CAR polypeptides used in the present invention can also bealtered as described below by using known methods. When one or morearbitrary amino acid residues in the amino acid sequence of a naturalCAR are replaced with other amino acid residues, substitution withconservative amino acid residues are preferred. Conservative amino acidresidues indicate amino acids comprising side chains similar to theamino acid residues prior to substitution. Amino acids can becategorized, for example, into the nine groups shown below, according tothe chemical properties of their side chains (hereinafter amino acidsare represented by one-letter symbols): (1) neutral hydrophobic sidechain (A, F, L, M, P, V, and W); (2) neutral polar side chain (C, G, N,Q, S, T, and Y); (3) basic side chain (H, K, and R); (4) acidic sidechain (D and E); (5) aliphatic side chain (A, G, I, L, and V); (6)aliphatic hydroxyl side chain (S and T); (7) amine-containing side chain(H, K, N, Q, and R); (8) aromatic side chain (F, W, and Y); and (9)sulfur-containing side chain (C and M). In addition, proteins in whichone or more amino acid residues in a natural CAR are modified byglycosylation, phosphorylation, or the like and proteins modified bydeglycosylation or dephosphorylation using chemical and enzymatictechniques, are also comprised in the altered CARs and altered CARpolypeptides of the present invention. Such altered CARs may also beused as CARs and CAR polypeptides in the present invention. Such proteinalterations and modifications can be carried out with the aim to improvethe stability and biological activity of CARs and CAR polypeptides. Bymeasuring binding activity to CARL, for example, such altered ormodified proteins can be confirmed as retaining their activities orotherwise. Herein, the phrase “retain biological activity” does notnecessarily mean the same activity level as that of a natural CAR, andthe activity may be higher or lower than the original activity.

The CARs and CAR polypeptides used in the present invention can beproduced as recombinant proteins as well as isolated from naturalsources as described above. “Recombinant CARs” and “recombinant CARpolypeptides” may be produced by expressing them as fusion proteins withmarker proteins for convenience of detection; the marker proteins canbe, for example, an enzyme such as alkaline phosphatase (SEAP) orβ-galactosidase; a binding protein such as maltose-binding protein orglutathione-S-transferase (GST); an Fc region of an antibody; or afluorescent protein such as green fluorescence protein. Examples ofparticularly preferred fusion proteins include those in which markerproteins are linked to an extracellular domain of a natural CAR.Recombinant proteins, comprising such fusion proteins, can be produced,for example, using appropriate expression systems, including in vitrosystems and host-vector systems.

Generally, expression vectors are first constructed to comprise chimericgenes comprising appropriate transcriptional and translationalregulatory regions and sequences encoding proteins of interest operablylinked to the regulatory regions. The transcriptional and translationalregulatory regions comprise DNA sequences recognized in selected hostsand required for the expression of the protein-coding sequences. SuchDNA sequences comprise, for example, promoters, enhancers,polyadenylation signals, operator sequences, ribosome binding sites,initiation signals, and terminators. When eukaryotic cells are used ashosts, expression vectors preferably comprise a promoter/enhancerelements. “Operably” linked or attached means that protein-codingsequences linked downstream of the regulatory regions are transcribedunder the regulation of the regulatory regions, without any shift in thereading frame, and expressed in hosts or to extracellular spaces.Appropriate expression vectors to be used for the recombinant CARpolypeptides include known expression vectors for mammalian cells,insect cells, plant cells, yeast cells, and bacterial cells.Alternatively, commercially available vectors may also be used.

Next, host cells are transformed with the constructed expressionvectors. Many cell lines have already been established as host celllines, and various transformation methods suited to such host cell lineshave also been established. Any of these known host cell lines may beused to produce recombinant CAR polypeptides, and those skilled in theart can carry out efficient transformation using appropriateintroduction methods suited to selected hosts. For example, suchtransformation methods include, but are not limited to, the followingmethods: transformation of prokaryotic cells can be carried out bycalcium treatment, electroporation, or the like; methods usingAgrobacterium and leaf disc methods are known for plant cells; andexamples for mammalian cells comprise calcium phosphate precipitation.Other known methods include nuclear microinjection, protoplast fusion,DEAE-dextran method, cell fusion, electroporation, lipofectamine method(GIBCO BRL), and methods using FuGENE6 reagent (Boehringer-Mannheim).For detailed information on mammalian cell transformation, reports ofKeown et al. ((1990) Methods in Enzymol. 185:527-537), Mansour et al.((1988) Nature 336:348-352), and such can be referred to. When naturalglycosylation is required, mammalian cells are preferably selected ashosts. Many cell lines, comprising A431, BHK, CHO, COS, CV-1, Hela,HL-60, Jurkat, 205, and 293 cells, are known as such mammalian cells,and these can be used to produce recombinant CAR polypeptides.

In the next step, desired proteins are expressed by culturingtransformed cells. Host cells are cultured by known methods suited tothe selected cells. For example, when mammalian cells are used as hosts,media such as Dulbecco's modified Eagle medium (DMEM; Virology 8:396(1959)), minimal essential medium (MEM; Science 122:501 (1952)),RPMI1640 (J. Am. Med. Assoc. 199:519 (1967)), 199 (Proc. Soc. Biol. Med.73:1 (1950)), Iscove's Modified Dulbecco's Medium (IMDM), or such areused, which can be supplemented as necessary with fetal calf serum (FCS)and such, and culture is carried out at a pH of about 6 to 8, at 30° C.to 40° C. for about 15 to 200 hours. The media may be changed, aerated,and stirred during culture, as required. Recombinant proteins can besecreted to extracellular spaces when secretory signals recognized inthe selected host cells are attached to the proteins. The expressedrecombinant proteins can be obtained from the host cells, or from theculture media when secreted.

Moreover, transgenic animals (see Susumu (1985) Nature 315:592-594;Lubon (1998) Biotechnol. Annu. Rev. 4: 1-54; and such), transgenicplants, or the like can be prepared as required and made to producerecombinant CAR polypeptides. When producing proteins using transgenicanimals, the proteins are preferably expressed in a tissue specificmanner (for example, in milk) by using promoters that ensuretissue-specific expression.

The CAR polypeptides used in the present invention may be purifiedproteins. For example, proteins expressed using recombination techniquescan be purified using conventional protein purification means. Knownprotein purification methods include chromatographies, such as affinity,ion exchange, gel filtration, reverse phase, adsorption, and hydrophobicinteraction, as well as methods such as ethanol precipitation,recrystallization, distillation, electrophoresis, dialysis,immunoprecipitation, solvent extraction, and ammonium sulfateprecipitation (Strategies for Protein Purification and Characterization:A Laboratory Course Manual, Marshak et al. ed., Cold Spring HarborLaboratory Press (1996)). Such methods can be used to purify CARpolypeptides. The CAR polypeptides used in the present invention may bepurified CAR polypeptides purified by such known means, or in some casesmay be partially purified proteins. Moreover, when for example CARpolypeptides are expressed as GST fusion proteins, purification methodsusing glutathione columns are effective. Meanwhile, nickel column-basedpurification methods can be used when CAR polypeptides are expressedwith attached histidine tags. When CAR polypeptides are produced as suchfusion proteins, unnecessary portions may be cleaved using enzymes suchas thrombin or factor Xa after purification if required.

CAR polypeptides used in the detection of Th1 cells in the presentinvention may be used bound onto carriers. The carriers for immobilizingmust have no adverse effect on the CAR polypeptides and cells. Suchcarriers include, for example, synthetic or natural organic polymercompounds; glasses; organic polymer materials, such as polystyrenes;inorganic materials, such as silica gels, alumina, and activatedcarbons; and such materials surface-coated with polysaccharides,synthetic polymers, and the like. The carrier shapes are notparticularly limited, and carriers of any shape can be used as long asthey do not impede contact between the polypeptides and cells. Examplescomprise membranous, fibrous, granular, hollow fiber-shaped, unwoven,porous, and honeycomb-shaped ones. The polypeptides can be bound, forexample, to the inner walls of reaction vessels, such as plates, dishes,and test tubes, and to beads and so on. The areas of contact betweenpolypeptides and cell samples can be controlled by altering thethickness, surface area, diameter, length, shape, and size of thecarriers used.

Detection of Th1 cells in the present invention is preferably carriedout using known methods after separating lymphocytes, preferably Tcells, and more preferably helper T cells from “cell samples comprisinglymphocytes”; or by detecting cells co-expressing lymphocyte markers,preferably co-expressing T cell markers, and more preferablyco-expressing helper T cell markers, for example CD4, in “cell samplescomprising lymphocytes”.

When CAR polypeptides are expressed as fusion proteins with markerproteins, methods corresponding to the adopted marker proteins are usedas the methods of the present invention for detecting Th1 cells. Forexample, when enzymes such as alkaline phosphatase or β-galactosidaseare used, detection is achieved based on the enzyme activity; whenbinding proteins such as glutathione-S-transferase or maltose-bindingprotein are used, detection is achieved based on the binding activity ofglutathione or maltose, respectively; when an Fc region of an antibodyis used, detection is achieved based on binding with an Fc-bindingprotein; and when a fluorescent protein such as green fluorescenceprotein is used, detection is achieved based on fluorescence.

Moreover, when not expressed as fusion proteins with marker proteins,CAR polypeptides bound to Th1 cells can be detected using antibodiesagainst CAR. When using anti-CAR antibodies in the detection, theantibodies used must recognize a part of the CAR polypeptide other thanthe part that binds to CARL on Th1 cells. Anti-CAR antibodies can beproduced by standard methods, in the same way as for the anti-CARLantibodies described in the next section, “Methods for detecting Th1cells using anti-CARL antibodies”. Specific examples of antibodyproduction comprise the method of Example 5. Alternatively, CARpolypeptides may be designed as fusion proteins with other polypeptidesrecognizable by antibodies, such that they can be detected by usingappropriate antibodies (see Examples 3 and 4). Commercially availableepitope-antibody systems can also be used (Experimental Medicine13:85-90 (1995)). CAR polypeptides may be produced as fusion proteinswith β-galactosidase, maltose-binding protein, glutathione-S-transferase(GST), green fluorescence protein (GFP), or the like, such thatdetection can be carried out without using a secondary antibody. Inaddition, small epitope-antibody systems, such as those ofpolyhistidine, influenza hemagglutinin HA, human c-myc, FLAG, and T7,are also known. When such tags are attached to CAR polypeptides, the CARpolypeptides can be detected using commercially available antibodiesagainst the tags. Alternatively, when labeled with radioisotopes, CARpolypeptides can be detected using scintillation counters. Moreover, theinteractions between CAR polypeptides and Th1 cells can be observed inreal time using biosensors that use surface plasmon resonance phenomenon(for example, BIAcore X (BIAcore)), without having to label thepolypeptides.

Further, for example, magnetic particles can be used as the carriers,and CAR polypeptides and CARL-expressing cells bound to the polypeptidescan be detected and collected using magnets. Magnetic devices for suchcollection have also been developed and can be used (for example, MACS(Daiichi Pure Chemicals Co.)). Moreover, Th1 cells expressing CARL canalso be selected by flow cytometry using cell sorters (FACS) and CARpolypeptides which have been labeled with fluorescence (for example,fluorescein isothiocyanate (FITC) and phycoerythrin) or the like.

Methods for Detecting Th1 Cells Using Anti-CARL Antibodies

The present inventors identified CARL as a CAR ligand on Th1 cells. Thepresent invention thus relates to methods for detecting Th1 cells usinganti-CARL antibodies. Specifically, Th1 cells can be detected by thesteps of:

-   (a) contacting anti-CARL antibodies with cell samples comprising    lymphocytes; and-   (b) detecting cells that bound to the anti-CARL antibodies in step    (a).

The “cell samples comprising lymphocytes” in the present methods are notparticularly limited as in the case of the above-described section“Methods for detecting Th1 cells using CAR”, and blood cell samplesexpected to contain lymphocytes, such as bone marrow and peripheralblood, can be used.

Moreover, cell samples can be contacted with antibodies in appropriatereaction solutions as in the case of the above-described CARpolypeptides.

Herein, “CARL” and “CARL polypeptide” include mouse proteins belongingto IgSF that comprise 379 residues comprising the amino acid sequence ofFIG. 7 (SEQ ID NO: 1; GenBank Accession No. AAH50133); their allelicvariants and splicing variants; and natural CARLs, such as proteinsderived from mammals other than mice and corresponding to theseproteins. Further, in addition to the above-described natural CARLs, the“CARL” and “CARL polypeptide” comprise non-natural polypeptidesfunctionally equivalent to these CARLs. For example, “CARL” and “CARLpolypeptide” comprise proteins with altered amino acid sequences fromnatural CARLs (altered CARLs) and CARLs produced as recombinant proteins(recombinant CARLs). In the present description, the terms “CARL” and“CARL polypeptide” are used interchangeably.

A cDNA sequence encoding the protein comprising the amino acid sequenceof SEQ ID NO: 1 is deposited under Accession No. BC050133 in GenBank(SEQ ID NO: 3). Moreover, a mouse protein in which 37 amino acids areinserted in place of the first 11 amino acids of BC050133, and thearginine at position 231 is replaced with glutamine, is also known as avariant (GenBank Accession No. XM_194453). Examples of human proteinscorresponding to these mice proteins comprise human AMICA protein(GenBank Accession No. AAN52117; SEQ ID NO: 2) encoded by a cDNAsequence of GenBank Accession No.AY138965 (SEQ ID NO: 4). CARL comprisesa signal sequence (the underlined portion in FIG. 7) predicted using theSignalIP program, and a transmembrane domain (portion underlined with adotted line in FIG. 7) predicted using the SMART program. Furthermore,the asparagine residue boxed in FIG. 7 was predicted to be glycosylatedby using the ScanProsite program. Also, the cysteine residues labeledwith an asterisk (*) in FIG. 7 were predicted to form disulfide bonds.The present invention showed that CARL was expressed selectively in Th1cells among lymphocytes, and that depending on the expression of CARL,CAR bound selectively to Th1 cells among lymphocytes. Proteins sharing abiological activity are known to be generally conserved evolutionarilyat their amino acid sequence level, as well as at the level of genesencoding the proteins. Therefore, examples of mammals, other than miceand humans, from which a “natural CARL” and a “natural CARL polypeptide”of the present invention are derived comprise rats, rabbits, dogs,horses, cats, pigs, bovine, goats, sheep, and primates, such as monkeys,gorillas, and chimpanzees. Allelic variants and the proteins derivedfrom these mammals can be obtained, for example, by obtaining cDNAs orgenes encoding the allelic variants or proteins from cDNA libraries,genomic libraries, or such using probes or primers produced based onknown nucleotide sequences or amino acid sequences of CARL describedabove and applying known techniques such as hybridization and PCR, thenexpressing these genes. Alternatively, CARL may also be prepared byconventional protein synthesis methods (for example, chemical synthesismethods and cell culture methods) based on known amino acid sequences.

In the present invention, “CARL” and “CARL polypeptide” refer to thefollowing proteins that bind to these animals' CAR polypeptides, whichare peptides encoded by the nucleotide sequence deposited in GenBankunder number BC050133 in the case of mice and peptides encoded by thenucleotide sequence deposited in GenBank under number AY138965 in caseof humans:

-   (1) proteins comprising the amino acid sequence of SEQ ID NO: 1    (mouse CARL);-   (2) proteins comprising an extracellular domain of the amino acid    sequence of the above (1);-   (3) proteins comprising the amino acid sequence of SEQ ID NO: 2    (human CARL);-   (4) proteins comprising an extracellular domain of the amino acid    sequence of the above (2);-   (5) proteins comprising the Ig-like domain 1 of the amino acid    sequences of the above (1) to (4);-   (6) proteins encoded by polynucleotides that hybridize under    stringent conditions to a cDNA sequence of SEQ ID NO: 3 (mouse cDNA)    or SEQ ID NO: 4 (human cDNA);-   (7) proteins comprising the amino acid sequences encoded by    polynucleotides that hybridize under stringent conditions to    polynucleotides encoding the amino acid sequence of a protein of the    above (1) to (5);-   (8) proteins comprising amino acid sequences with 90% or higher    sequence homology to the amino acid sequence of a protein of the    above (1) to (5); and-   (9) proteins comprising amino acid sequences with a deletion,    substitution, addition, or insertion of one or more amino acid    residues in the amino acid sequence of a protein of the above (1) to    (5).

Genes encoding CARLs exhibiting biological activities equivalent tothose of a CARL of SEQ ID NO: 1 or 2 can be obtained by hybridization byreferring to the explanation on the hybridization conditions forobtaining CAR polypeptides described in the above section “Methods fordetecting Th1 cells using CAR”. Amino acid sequences with a deletion,substitution, addition, or insertion of one or more amino acid residuesand exhibiting biological activities equivalent to those of a CARL ofSEQ ID NO: 1 or 2 can be obtained by referring to the explanation of themethods for deleting, adding, substituting, or inserting amino acidresidues for obtaining CAR polypeptides described in the above section“Methods for detecting Th1 cells using CAR”.

The anti-CARL antibodies of the present invention used for detecting Th1cells may be any antibodies as long as they can bind to CARL on Th1cells, and comprise polyclonal antibodies, monoclonal antibodies,chimeric antibodies, single-chain antibodies (scFV; Huston et al. (1988)Proc. Natl. Acad. Sci. USA 85: 5879-5883; The Pharmacology of MonoclonalAntibody, Vol. 113, Rosenburg and Moore ed., Springer Verlag (1994) p.269-315), humanized antibodies, multispecific antibodies (LeDoussal etal. (1992) Int.J.Cancer Suppl. 7:58-62; Paulus (1985) Behring Inst.Mitt. 78:118-132; Millstein and Cuello (1983) Nature 305:537-539;Zimmermann (1986) Rev. Physiol. Biochem. Pharmacol. 105: 176-260; VanDijk et al. (1989) Int.J.Cancer 43: 944-949), and antibody fragmentscomprising antigen-binding sites, such as Fab, Fab′, F(ab′)2, and Fv.Further, the anti-CARL antibodies may be modified by PEG or the like, asrequired. Alternatively, the antibodies may be produced as fusionproteins with β-galactosidase, maltose-binding protein, GST, greenfluorescence protein (GFP), or the like, such that detection can becarried out without using a secondary antibody. Also, by labeling theantibodies with biotin or the like, the antibodies can be collectedusing avidin, streptavidin, or such.

The anti-CARL antibodies can be produced using CARL, fragments thereof,or cells expressing any one of these, as sensitizing antigens. When aCARL fragment is used as an antigen, a CARL extracellular region ispreferably used. If required (for example, when using a short antigenfragment), the fragment may be conjugated with carriers, such as bovineserum albumin, keyhole limpet hemocyanin, and ovalbumin, and then usedas an immunogen. Furthermore, if required, known adjuvants, such asaluminium adjuvant, Freund's complete or incomplete adjuvants, orpertussis adjuvant, can be used to enhance the immune response againstsuch antigens.

For example, mammals can be immunized with CARL or fragments thereof,together with an adjuvant as required, to obtain polyclonal antibodiesagainst CARL as sera from the animals. Animals of Rodentia, Lagomorpha,and Primates are typically used as immunized mammals herein, althoughthey are not limited thereto. Examples comprise Rodentia such as mice,rats, and hamsters; Lagomorpha such as rabbits; Primates, comprisingmonkeys such as crab-eating macaques, Rhesus monkeys, baboons, andchimpanzees. Immunization of the animals is carried out by:appropriately diluting and suspending a sensitizing antigen in PBS orphysiological saline; further adding an adjuvant and emulsifying ifrequired; and then injecting intraperitoneally, subcutaneously, or intothe footpad. Then, a sensitizing antigen combined with Freund'sincomplete adjuvant is preferably administered several times every fourto 21 days. Antibody production can be verified by measuring antibodylevels in the sera of the immunized animals. The obtained seracomprising the antibodies may be further purified, if required.

Meanwhile, monoclonal antibodies can be produced by the followingprocedure: first, the spleen are excised from animals immunized by themethod described above. Immune cells are separated from the spleen andfused with appropriate myeloma cells using polyethylene glycol or suchto produce hybridomas. Regarding cell fusion, reports such as that ofGalfre and Milstein ((1981) Methods Enzymol. 73:3-46) can be referredto. Herein, examples of particularly suited myeloma cells comprise cellsthat allow drug selection of fused cells. When such myeloma enablingdrug selection are used, cells are cultured in a culture medium in whichcells other than fused cells are killed (such as HAT medium) and fusedcells are selectively collected. Then, clones producing antibodies thatbind to CAR are selected from the produced hybridomas. The selectedclones are transplanted in the peritoneal cavities of mice or such, andascites fluid can be collected from the animals to obtain monoclonalantibodies.

Hybridomas can also be obtained by first using an immunogen to sensitizehuman lymphocytes that have been infected by EB virus in vitro, thenfusing the sensitized lymphocytes with human-derived myeloma cells (suchas U266) to obtain hybridomas that produce human antibodies (JapanesePatent Application Kokai Publication No. (JP-A) S63-17688 (unexamined,published Japanese patent application)). In addition, human antibodiescan also be obtained by using antibody-producing cells generated bysensitizing transgenic animals which have the repertoire of humanantibody genes (WO 92/03918; WO 93/02227; WO 94/02602; WO 94/25585; WO96/33735; WO 96/34096; Mendez et al. (1997) Nat. Genet. 15: 146-156,etc.). Methods that do not use hybridomas are exemplified by methods inwhich antibodies are produced by introducing cancer genes to immortalizeimmune cells, such as antibody-producing lymphocytes.

The present invention also comprises human monoclonal antibodies. Themonoclonal antibodies can be produced by immunizing humanantibody-producing nonhuman transgenic mammals, such as humanantibody-producing transgenic mice, with immunogens (antigens), such asCARL or fragments thereof, or cells expressing any of these, and thenproducing monoclonal antibodies according to existing conventionalmethods for producing monoclonal antibodies.

Specifically, for example, nonhuman transgenic mammals producing humanantibodies are immunized with an antigen, in combination with Freund'sadjuvant if required. Polyclonal antibodies can be prepared from seraobtained from the immunized animals. Moreover, monoclonal antibodies canbe produced by preparing fused cells (hybridomas) fromantibody-producing cells obtained from the immunized animals and myelomacells that lack the ability to secrete endogenous immunoglobulin,cloning the hybridomas, then selecting clones producing monoclonalantibodies that exhibit specific affinity for the antigen used toimmunize the mammals.

More specifically, the antibodies can be produced as described below.Specifically, the nonhuman transgenic mammals producing human antibodies(particularly preferred are the “human antibody-producing transgenicmice” described below) are immunized with a desired antigen, incombination with Freund's adjuvant if required, by injecting ortransplanting the antigen once or several times subcutaneously,intramuscularly, intravenously, intraperitoneally, or into footpads.Typically, immunization is carried out one to four times approximatelyevery one to 14 days from the first immunization, and antibody-producingcells are obtained from the immunized mammals approximately one to fivedays after the final immunization. The frequency and time intervals ofimmunization can be appropriately varied depending on the properties ofthe antigens being used, or the like.

Fused cells (hybridomas) that secrete human monoclonal antibodies can beprepared by the method of Köhler and Milstein (Nature, Vol. 256, p.495-497, 1975) or modified methods based on this method. Specifically,they are prepared by fusing antibody-producing cells from the spleen,lymph nodes, bone marrow, tonsilla, or such, preferably from the spleen,of human antibody-producing nonhuman transgenic mammals immunized asdescribed above, and myeloma cells lacking the ability to secreteendogenous immunoglobulin, which are derived from mammals such aspreferably mice, rats, guinea pigs, hamsters, rabbits, or humans, morepreferably mice, rats, or humans.

Myeloma cells that can be used in cell fusion include, for example, themouse-derived myelomas P3/X63-AG8.653 (ATCC No. CRL-1580),P3/NSI/1-Ag4-1 (NS-1), P3/X63-Ag8.U1 (P3U1), SP2/0-Ag14 (Sp2/O, Sp2),NS0, PAI, F0, and BW5147; the rat-derived myelomas 210RCY3-Ag.2.3; thehuman-derived myelomas U-266AR1, GM1500-6TG-A1-2, UC729-6, CEM-AGR,D1R11, and CEM-T15. Screening for cells producing monoclonal antibodies(for example, hybridomas) can be performed, for example, by culturingcells in microtiter plates, then measuring the reactivity of culturesupernatants from wells in which proliferation was observed to theaforementioned immunogen used for the immunization, by using, forexample, enzyme immunoassays such as RIA (radioimmunoassay) and ELISA(enzyme-linked immunosorbent assay).

Monoclonal antibodies can be produced from hybridomas in vitro, or invivo in the ascites fluid and such of mice, rats, guinea pigs, hamsters,rabbits, and the like, preferably of mice or rats, and more preferablyof mice, and isolated from the obtained culture supernatants or from theascites fluid of the mammals.

Moreover, the monoclonal antibodies of the present invention can beproduced on a large scale by using the method described below.

-   (1) Genes (cDNAs or the like) encoding the heavy and light chains of    the monoclonal antibodies are cloned from the hybridomas.-   (2) Expression vectors are prepared by inserting the cloned genes    encoding the heavy and light chains each into a different or a same    vector.-   (3) The vectors are introduced into fertilized eggs of desired    nonhuman mammals (such as goats).-   (4) The fertilized eggs into which the genes have been introduced    are transplanted in the uteri of foster mothers to obtain nonhuman    chimeric animals.-   (5) The chimeric goats are further crossed with other nonhuman    mammals to create transgenic nonhuman mammals (bovine, goats, sheep,    pigs, or such) in which the genes encoding the heavy and light    chains are integrated in the endogenous genes.-   (6) The monoclonal antibodies derived from the human monoclonal    antibody genes are obtained on a large scale from milk of the    nonhuman transgenic mammals (Nikkei Science, April 1997, p. 78-84).

Human monoclonal antibodies produced by this method are also included inthe present invention. When cells producing the monoclonal antibodiesare cultured in vitro, it is possible to use any known nutrition mediaor nutrition media prepared based on known basal media that is used togrow, maintain, and preserve hybridomas and to produce monoclonalantibodies in culture supernatants, according to various criteria, suchas the characteristics of the cell types to be cultured, the purpose ofthe experimental studies, and the culture methods.

Basal media include, for example, low calcium media such as Ham's F12medium, MCDB153 medium, and low calcium MEM; and high calcium media suchas MCDB104 medium, MEM, D-MEM, RPMI1640 medium, ASF104 medium, and RDmedium. The basal media can contain, for example, sera, hormones,cytokines, and/or various inorganic or organic substances depending onthe purposes.

Monoclonal antibodies can be purified or isolated by subjecting theculture supernatants or ascites fluid described above to saturatedammonium sulfate, euglobulin precipitation methods, caproic acidmethods, caprylic acid methods, ion exchange chromatography (DEAE, DE52,and the like), or affinity column chromatography such asanti-immunoglobulin columns or protein A columns.

The human monoclonal antibodies of the present invention also includehuman monoclonal antibodies comprising heavy and/or light chains thatcomprise amino acid sequences with a deletion, substitution, or additionof one or more amino acids in the amino acid sequences of the heavyand/or light chains constituting the antibodies.

The anti-CARL antibodies further comprise antibody fragments, asdescribed above. Antibody fragments can be produced by treating thepolyclonal or monoclonal antibodies with enzymes such as papain orpepsin. Alternatively, they can be produced by genetic engineeringtechniques using a gene that encodes an antibody fragment (see Co etal., (1994) J. Immunol. 152: 2968-2976; Better and Horwitz (1989)Methods Enzymol. 178: 476-496; Pluckthun and Skerra (1989) MethodsEnzymol. 178: 497-515; Lamoyi (1986) Methods Enzymol. 121: 652-663;Rousseaux et al. (1986) 121: 663-669; Bird and Walker (1991) TrendsBiotechnol. 9: 132-137).

Multispecific antibodies comprised in the anti-CARL antibodies includebispecific antibodies (BsAb), diabodies (Db), and such. Multispecificantibodies can be produced by methods such as: (1) chemically couplingantibodies having different specificities with different types ofbifunctional linkers (Paulus (1985) Behring Inst. Mitt. 78: 118-132);(2) fusing hybridomas that secrete different monoclonal antibodies andmaking these hybridoma produce BsAb (Millstein and Cuello (1983) Nature305: 537-539); or (3) transfecting eukaryotic cell expression systems,such as mouse myeloma cells, with a light chain gene and a heavy chaingene of different monoclonal antibodies (four types of DNA in total),followed by the isolation of a bispecific monovalent portion (Zimmermann(1986) Rev. Physio. Biochem. Pharmacol. 105: 176-260; Van Dijk et al.(1989) Int. J. Cancer 43: 944-949). On the other hand, diabodies aredimer antibody fragments comprising two bivalent polypeptide chains thatcan be constructed by gene fusion. These can be produced using knownmethods (see Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; EP404097; WO 93/11161).

The antibodies and antibody fragments can be recovered and purified, notonly using protein A or G, but also by using methods similar to thosecommonly used to purify other proteins (Antibodies: A Laboratory Manual,Harlow and David Lane ed., Cold Spring Harbor Laboratory Press (1988)).For example, when protein A is used to purify the antibodies of thepresent invention, known protein A columns, such as Hyper D, POROS, andSepharose F. F. (Pharmacia) can be used. The concentrations of theobtained antibodies can be determined by measuring the absorbance ofsamples or the antigen binding activities of antibodies in samples.

The antigen binding activities of antibodies can be measured byabsorbance measurement, fluorescence antibody methods, enzymeimmunoassays (EIA), radioimmunoassays (RIA), enzyme-linked immunosorbentassays (ELISA), or such. When measurements by ELISA are carried out,samples comprising an antibody of interest are added after the protein(a CAR or CARL or portion thereof) is immobilized. Herein, the samplescomprising antibodies include culture supernatants of antibody-producingcells and purified antibodies. Next, a secondary antibody thatrecognizes anti-CARL antibodies is added and the plates are incubated.The plates are then washed, and the label attached to the secondaryantibody is detected. Specifically, when the secondary antibody islabeled, for example, with alkaline phosphatase, the antigen bindingactivity can be measured by adding an enzyme substrate, such asp-nitrophenyl phosphate, and measuring the absorbance. Moreover,antibody activity may be evaluated using commercially available systems,such as BIAcore (Amersham Pharmacia).

The detection of Th1 cells using anti-CARL antibodies of the presentinvention is preferably carried out after using known methods toseparate lymphocytes, preferably T cells, and more preferably helper Tcells from “cell samples comprising lymphocytes”; or by detecting in“cell samples comprising lymphocytes” those cells co-expressinglymphocyte markers, preferably cells co-expressing T cell markers, andmore cells preferably co-expressing helper T cell markers, for exampleCD4.

In the methods of the present invention for detecting Th1 cells usinganti-CARL antibodies, the antibodies may be immobilized on appropriatecarriers before contacting with cells. Alternatively, cell samples canalso be contacted with antibodies under conditions that allow thebinding of CARL-expressing cells to anti-CARL antibodies, and then cellsthat bound to the antibodies can be selectively detected or recoveredthrough purification using the affinity of the antibodies. For example,when the anti-CARL antibodies are bound to biotin, cell-antibodycomplexes can be purified through a contact with plates, columns, andsuch to which avidin or streptavidin are bound. Moreover, the anti-CARLantibodies may be immobilized on any of the carriers exemplified abovefor immobilization of CAR.

More specifically, methods for detecting Th1 cells using the anti-CARLantibodies include fluorescence antibody methods (see MonoclonalAntibodies: Principle and Practice, 3rd ed., Academic Press (1996)),ELISA, RIA, immunohistochemical staining, comprising immunohistologicalstaining and immunocytological staining (for example, ABC and CSAmethods; see Monoclonal Antibodies: Principle and Practice, 3rd ed.,Academic Press (1996)), Western blotting, and immunoprecipitation. Whenusing ELISA, substances that can easily detect antibodies are used assubstrates, or enzymes (for example, peroxidases) that producedetectable products are used as labels and substrates are reactedthereto, and the concentration of the substrates or products is measuredusing spectrophotometers or the like. ELISA also includes sandwichELISA. In this method, two types of antibodies that bind to differentantigenic sites are used, and analysis is carried out by labeling oneantibody with an enzyme or such. In RIA, the antibodies areradiolabeled. The labeled antibodies can be detected using scintillationcounters or the like. In immunohistochemical staining, antibodies arelabeled and reacted to tissues or cells, and then labels are detectedusing microscopes. Fluorescent substances and enzymes catalyzingreactions that produce a dye are used as labels. In immunoprecipitation,antibodies and cell samples are reacted, and then carriers that bindspecifically to immunoglobulins (protein G-Sepharose or the like) areadded to the reaction solution to precipitate the cell-antibodycomplexes.

Moreover, Th1 cells can be detected by detecting the anti-CARLantibodies in the same way as when using CAR polypeptides. Th1 cellsexpressing CARL can also be selected by, for example, flow cytometryusing cell sorters and anti-CARL antibodies labeled with fluorescence orsuch, or by using biosensors that use the surface plasmon resonancephenomenon. Flow cytometry and methods using magnets as carriers areparticularly simple techniques, and are preferably used in the detectionof Th1 cells of the present invention.

Kits for Detecting Th1 Cells

The CAR polypeptides and anti-CARL antibodies described above can becomprised in kits for detecting Th1 cells. The present invention thusrelates to kits for detecting Th1 cells, which comprise the CARpolypeptides or anti-CARL antibodies. The CAR polypeptides and anti-CARLantibodies may be comprised in the kits, immobilized on carriers.

In addition to the CAR polypeptides or anti-CARL antibodies, othermaterials required to detect Th1 cells can be combined in the kits ofthe present invention. The kits of the present invention can comprise,for example, reagents, containers, devices, and such required to detectthe polypeptides and antibodies. The CAR polypeptides or anti-CARLantibodies comprised in the kits, and the other necessary materials maybe packed together or separately, or a portion may be packed together.The shape of the packaging is not limited.

The materials required for the detection include, for example, buffersor the like to dilute the polypeptides, antibodies, or cell samples. Inaddition, the kits may comprise, for example, anti-CAR antibodies fordetecting the polypeptides. The kits may also comprise secondaryantibodies for detecting antibodies, and when the antibodies are labeledwith an enzyme, they may also comprise substrates or the like for thereactions catalyzed by the enzyme. When the polypeptides or antibodiesare bound to magnetic particles, magnets and such may be included in thekits. An instruction manual is preferably attached to the kits,describing in detail the detection procedures using the CAR polypeptidesor anti-CARL antibodies comprised in the kits of the present inventionfor detecting Th1 cells. Such instruction manuals can be comprised aspamphlets in the kits, or the instructions may be printed on thepackages or such of the kits.

Methods for Examining the Ratio between Th1 and Th2 Cells

The aforementioned methods for detecting Th1 cells using CAR oranti-CARL antibodies and kits for detecting Th1 cells can be used toexamine the ratio between Th1 and Th2 cells in biological samples. Thus,the present invention also relates to methods for examining the ratiobetween Th1 and Th2 cells. Specifically, helper T cells can bespecifically detected in cell samples comprising lymphocytes (forexample, using CD4 as a marker) and furthermore, Th1 cells can bedetected by the above-described methods for detecting Th1 cells, whichuse CAR or anti-CARL antibodies. Ratios between Th1 and Th2 cells incell samples examined as above can be used for diagnosing diseasesinvolving a selective imbalance of Th1 or Th2 cells. For example, it isthought that the Th1 response is dominant in organ-specific autoimmunediseases, while the Th2 response is dominant in cancers, allergies,parasite infections, and the like, and these diseases can be diagnosedusing the methods of the present invention for examining the ratiobetween Th1 and Th2 cells.

Examples of diseases particularly preferably diagnosed by the methods ofthe present invention for examining the ratio between Th1 and Th2 cellscomprise atopic diseases. The present invention thus relates to methodsfor diagnosing subjects for atopic diseases, based on the ratio betweenTh1 and Th2 cells determined by methods of the present invention forexamining the ratio between Th1 and Th2 cells. Atopic diseases diagnosedby the present methods comprise bronchial asthma, allergic rhinitis, andatopic dermatitis.

Th2 cells are known to be more dominant in such atopic diseases than Th1cells (Okazaki H. et al. (2002) Clin Exp Allergy. 32(8):1236-1242; andKim J. H. et al. (2004) J Asthma. 41(8):869-876). Thus, atopicdermatitis can be diagnosed by examining the ratio between Th1 and Th2cells.

Method of Screening for CAR-CARL Binding Inhibitors

The present invention further provides methods of screening forinhibitors of the binding between CAR and CARL (CAR-CARL bindinginhibitors). Specifically, CAR-CARL binding inhibitors are screened bythe steps of: (a) contacting CAR and CARL in the presence of testsubstances; (b) detecting the binding between CAR and CARL in step (a);(c) comparing the degree of the detected binding between CAR and CARLwith that in the absence of the test substances; and (d) selecting testsubstances that suppress the binding between CAR and CARL, as comparedto in the absence of the test substance, as inhibitors of the bindingbetween CAR and CARL.

Inhibitors of the binding between CAR and CARL are not limited toparticular types of substances. Thus, the test substances to be used inthe screening methods of the present invention may be any substances.Examples comprise expression products of gene libraries, libraries ofsynthetic low-molecular-weight compounds, synthetic peptide libraries,antibodies, substances released from bacteria, cell (microorganisms,plant cells, and animal cells) extracts and culture supernatants,purified or partially purified polypeptides, marine organisms, extractsderived from plants or animals, soil, and random phage peptide displaylibraries. Inhibitors screened by the present methods inhibit thebinding between CAR and CARL, and consequently inhibit adhesion betweenactivated Th1 cells expressing CARL, and epithelial and endothelialcells expressing CAR. Such inhibitors are thus expected to suppress theadhesion of Th1 cells and thereby to alleviate the conditions indiseases such as rheumatoid arthritis, in which the action of Th1 cellsis a factor. Therefore, inhibitors obtained by the present screening canbe candidates for therapeutic or preventive agents for diseases in whichTh1 cells are a factor.

The CAR and CAR polypeptides used herein are those described in theabove section “Methods for detecting Th1 cells using CAR”. Natural CARs,as well as their fragments, altered forms, modified forms and the like,which retain CARL-binding activity, can be used. The CAR and CARpolypeptides may be produced by expressing them as fusion proteins withmarker proteins for convenience of detection, for example enzymes suchas alkaline phosphatase (SEAP) and β-galactosidase; binding proteinssuch as glutathione-S-transferase (GST) and maltose-binding protein; Fcregions of antibodies; or fluorescent proteins such as greenfluorescence protein.

Meanwhile, similarly to CAR and CAR polypeptides, the “CARL” and “CARLpolypeptides” used in the present invention comprise natural CARLs aswell as proteins comprising amino acid sequences altered from naturalCARLs, as described in the above section “Methods for detecting Th1cells using anti-CARL antibodies”. CARL polypeptides used in the presentmethods may be any CARL polypeptides, as long as they retain bindingactivity to CAR, so they can be used in screening for inhibitors ofCAR-CARL binding and are preferably proteins comprising a CARLextracellular domain. Furthermore, examples of polypeptides functionallyequivalent to CARL include polypeptides comprising the CAR-bindingdomain of natural CARL, domain 1 in particular, and proteins exhibitinghigh amino acid sequence homology to natural CARL, particularly in theiractive sites. Herein domain 1 is a domain predicted from homology toIg-like domains, and is, for example, the region comprising the aminoacid sequence from position 30 to 139 in SEQ ID NO: 1 and the regioncomprising the amino acid sequence from position 27 to 136 in SEQ ID NO:2. Protein domains can be predicted at domain searching sites, forexample, on the world wide web at smart.embl-heidelberg.de/. HumanAMICA, which corresponds to mouse CARL that binds to CAR, had a homologyof 37.4% over its entire amino acid sequence. In the present invention,thus, high homology in the amino acid sequence, means an identity of,for example, 30%, 35%, 40%, or 50% or more, preferably 60% or more, morepreferably 70% or more (for example, 80%, 90%, or 95% or more).

In the first step (a), CAR and CARL, which bind each other, arecontacted in the presence of a test substance. This contact is typicallycarried out by adding CARL to a reaction solution comprising CAR and atest substance, or by adding CAR to a reaction solution comprising CARLand a test substance. Reaction solutions used herein are notparticularly limited, and any reaction solution may be used, as long asit does not inhibit the reaction between CAR and CARL. Furthermore, theduration of such contact is not particularly limited, and the contactcan be long enough for the binding between CAR and CARL, for example,for one, two, three or five minutes or more. Conditions for the contactcan be appropriately determined with reference to Example 11, whichinvestigates the affinity between CAR and the CARL extracellular region.

Herein, screening may be carried out with either CAR or CARL bound ontoa carrier. The various carriers exemplified in the above section“Methods for detecting Th1 cells using CAR” can be used as the carriers.When one of the polypeptides is immobilized on a carrier, inhibitors arescreened, for example, as follows: first, CAR and CARL are contacted inthe presence of a test compound. In this example, CAR is assumed to beimmobilized. After contact, the carriers are washed thoroughly to washaway CARL that did not bind with CAR. Then, CARL that bound to CAR isdetected. CARL can be detected using antibodies against CARL.Alternatively, when labeled CARL is used, the labels can be detectedappropriately.

Furthermore, cells expressing either CAR or CARL may be used as a CAR orCARL of the present invention. Such cells may be appropriate host cellsinto which genes encoding CAR or CARL have been introduced and areexpressed on the cell membrane.

Further, CAR-CARL binding inhibitors can be screened using thetwo-hybrid method (Dalton and Treisman (1992) Cell 68:597-612; Fieldsand Sternglanz (1994) Trends Genet. 10:286-292). The screening can beperformed, for example, using commercially available systems, such asthe MATCHMAKER Two-Hybrid system, Mammalian MATCHMAKER Two-Hybrid AssayKit, and MATCHMAKER One-Hybrid system from Clontech; and the HybriZAPTwo-Hybrid Vector System from Stratagene. The two-hybrid method detectsthe interaction between two types of proteins in vivo using thetranscriptional activator GAL4 of a Saccharomyces yeast. GAL4 comprisesa DNA binding domain and a transcriptional activation domain, andactivates transcription through binding to UAS_(G), an upstream GALactivation sequence in yeast. Given this, expression vectors encodingfusion proteins are constructed, in which either one of CAR or CARL isfused to the DNA binding domain and the other one is fused to thetranscriptional activation domain. Further, an expression vector for areporter gene operably linked with a promoter comprising the activationsequence UAS_(G) is prepared, and introduced into host cells along withthe above expression vectors for the two fusion proteins. The reportergene is expressed when the CAR and CARL moieties in the two types offusion protein bind, and whether CAR and CARL bound can be determinedbased on this expression. At this time, by including a test substance inthe system, inhibition by the test substance of binding between CAR andCARL can be assessed. If a test substance can be encoded by a gene, itmay be introduced into host cells as an expression vector constructed toexpress the gene encoding the substance. Various reporter genes areknown and, for example, the Ade2 gene, lacZ gene, CAT gene, luciferasegene, HIS3 gene, β-galactosidase, and β-lactamase can be used.

The interaction between CAR and CARL can be observed in real time usinga biosensor which uses the surface plasmon resonance phenomenon (forexample, BIAcore (Amersham Pharmacia)), without having to label thepolypeptides. Thus, whether a test substance acts as an inhibitor of thebinding between CAR and CARL can be detected using such biosensor, byadding the test substance at the time CAR and CARL are contacted. Forspecific methods Example 11 can be referred to, for example.

CAR-CARL Binding-Inhibiting Antibodies

Furthermore, the present invention relates to antibodies that inhibitthe binding between CAR and CARL. The antibodies of the presentinvention comprise polyclonal antibodies, monoclonal antibodies,chimeric antibodies, single-chain antibodies (scFV; Huston et al. (1988)Proc. Natl. Acad. Sci. USA 85: 5879-5883; The Pharmacology of MonoclonalAntibody, Vol. 113, Rosenburg and Moore ed., Springer Verlag (1994) p.269-315), humanized antibodies, multispecific antibodies (LeDoussal etal. (1992) Int.J.Cancer Suppl. 7:58-62; Paulus (1985) Behring Inst.Mitt. 78:118-132; Millstein and Cuello (1983) Nature 305:537-539;Zimmermann (1986) Rev. Physiol. Biochem. Pharmacol. 105: 176-260; VanDijk et al. (1989) Int.J.Cancer 43: 944-949), and antibody fragmentscomprising antigen-binding sites, such as Fab, Fab′, F(ab′)2, and Fv.Further, the antibodies of the present invention may be modified by PEGor the like, as required. Alternatively, the antibodies of the presentinvention may be produced as fusion proteins with β-galactosidase,maltose-binding protein, GST, green fluorescence protein (GFP), or thelike, such that detection can be carried out without using a secondaryantibody. Also, by labeling the antibodies with biotin or the like, theantibodies can be collected using avidin, streptavidin, or such.

The antibodies of the present invention can be produced using a CAR orfragments thereof, a CARL or fragments thereof, or cells expressing anyone of these as sensitizing antigens, by the same method as that usedfor the anti-CARL antibodies, which is described in the above section“Methods for detecting Th1 cells using anti-CARL antibodies”. In apreferred embodiment, the antibodies of the present invention areanti-CAR antibodies or anti-CARL antibodies. The antibodies of thepresent invention may be, for example, anti-CAR antibodies produced bythe hybridomas: @mCAR:5E9-1-1 internationally deposited under AccessionNumber: FERM BP-10317; @mCAR:5G11-1-1-11 internationally deposited underAccession Number: FERM BP-10318; and @mCAR:4C9-1-1 internationallydeposited under Accession Number: FERM BP-10320. Furthermore, theantibodies of the present invention may also be the anti-CARL antibodiesproduced by hybridoma @mCARL:#3.11 internationally deposited underAccession Number: FERM BP-10319. However, the antibodies of the presentinvention are not limited to the antibodies described above. Asdisclosed in detail in the Examples below, for example, the monoclonalantibodies produced by clone 5G11 obtained by the method described inExample 5 inhibit the binding between CAR and Th1 cells. Further, #3antibody obtained by the method described in Example 14 inhibits theadhesion between CARL and cells expressing CAR, and Example 17demonstrates that #3 antibody has an effect in the treatment of contactdermatitis. Example 16 shows that monoclonal antibodies #5, #49, and #77against human CARL inhibit the adhesion between human CARL and cellsexpressing human CAR.

Hybridomas 5E9, 5G11 and 4C9, obtained in Example 5, and hybridoma #3,obtained in Example 14, were deposited as described below. In thepresent description, 5E9 is identical to @mCAR:5E9-1-1 described below;5G11 is identical to @mCAR:5G11-1-1-11 described below; #3 is identicalto @mCARL:#3.11 described below; and 4C9 is identical to @mCAR:4C9-1-1described below. Antibodies produced by these hybridomas are called bythe same names as the corresponding hybridomas.

@mCAR:5E9-1-1

-   (1) Name and address of depositary institution    Name: National Institute of Advanced Industrial Science and    Technology, International Patent Organism Depositary    Address: (Postal code: 305-8566) Central 6, 1-1-1 Higashi,    Tsukuba-shi, Ibaraki-ken, Japan-   (2) Date of international deposit: Apr. 12, 2005 (Date of domestic    deposit: Apr. 27, 2004)-   (3) International Accession Number: FERM BP-10317    (Domestic Accession Number: P-20031; Domestic Acceptance Number:    FERM AP-20031)

@mCAR:5G11-1-1-11

-   (1) Name and address of depositary institution    Name: National Institute of Advanced Industrial Science and    Technology, International Patent Organism Depositary    Address: (Postal code: 305-8566) Central 6, 1-1-1 Higashi,    Tsukuba-shi, Ibaraki-ken, Japan-   (2) Date of international deposit: Apr. 12, 2005 (Date of domestic    deposit: Apr. 27, 2004)-   (3) International Accession Number: PERM BP-10318    (Domestic Accession Number: P-20032; Domestic Acceptance Number:    FERM AP-20032)

@mCARL:#3.11

-   (1) Name and address of depositary institution    Name: National Institute of Advanced Industrial Science and    Technology, International Patent Organism Depositary    Address: (Postal code: 305-8566) Central 6, 1-1-1 Higashi,    Tsukuba-shi, Ibaraki-ken, Japan-   (2) Date of international deposit: Apr. 12, 2005-   (3) International Accession Number: FERM BP-10319

@mCAR:4C9-1-1

-   (1) Name and address of depositary institution    Name: National Institute of Advanced Industrial Science and    Technology, International Patent Organism Depositary    Address: (Postal code: 305-8566) Central 6, 1-1-1 Higashi,    Tsukuba-shi, Ibaraki-ken, Japan-   (2) Date of international deposit: Apr. 12, 2005-   (3) International Accession Number: FERM BP-10320

Since the antibodies of the present invention inhibit the bindingbetween CAR and CARL, they are expected to suppress the adhesion of Th1cells and cell adhesion during inflammation, and thus to alleviatediseases. Further, because the binding between CAR and CARL may beinvolved in the infiltration of Th1 cells, they are expected to beuseful in elucidating the mechanism of cell infiltration.

In particular, of the antibodies of the present invention, antibodiesprepared using CAR or fragments thereof as antigens can be used topurify CAR or fragments thereof, in the same way as for genericantibodies. They can also be used in drug delivery systems targetingCAR.

Antibodies produced using CARL or fragments thereof as antigens can beused to purify CARL or its fragments, and can be used in drug deliverysystems targeting CARL, in the same way as for CAR. Meanwhile, as forthe CAR-CARL binding inhibitors described above, the antibodies areexpected to suppress the adhesion of Th1 cells in diseases in which theaction of Th1 cells is a factor, such as rheumatoid arthritis, and thusto alleviate diseases. These antibodies of the present invention can becandidates for therapeutic or preventive agents for diseases in whichTh1 cells are a factor. In fact, the present invention demonstrates thatanti-CARL antibodies have an effect in the treatment of contactdermatitis (Example 17).

Compositions Comprising Anti-CAR Antibodies and Compositions ComprisingAnti-CARL Antibodies

As described above, the antibodies of the present invention inhibitbinding between CAR and CARL, and thus can be used to inhibit theadhesion of activated Th1 cells expressing CARL to cells expressing CAR,such as epithelial cells and endothelial cells. Given this, the presentinvention relates to cell adhesion inhibitors comprising the antibodiesof the present invention. Such compositions can be used when examiningthe binding abilities of CAR and CARL. The inhibitors of the presentinvention are, for example, the antibodies of the present inventiondissolved in appropriate buffers or the like. Further, as required,preservatives, antiseptics, stabilizers, and such can be added withinranges that do not influence the antibody activities.

Furthermore, since the antibodies of the present invention suppress theadhesion of Th1 cells, they are expected to suppress cell infiltrationduring inflammation, and to be able to alleviate diseases. Thus,compositions comprising such antibodies are expected to be usable astherapeutic or preventive agents for diseases in which the action of Th1cells is a factor. For example, compositions comprising the antibodiesof the present invention can be used as therapeutic or preventive agentsfor contact dermatitis. In case the antibodies are used as suchtherapeutic or preventive agents, considering the immunogenicity, it isdesirable to use human antibodies or humanized antibodies when thetherapeutic or preventive agents are used for humans.

The antibodies can be formulated in consideration of their propertiesbased on known methods for producing antibody preparations. When suchcompositions of the present invention are used as therapeutic orpreventive agents, the compositions comprise the antibodies of thepresent invention as active ingredients, and physiologically acceptablecarriers, excipients, diluents, and such are appropriately mixed in. Themode of administration can be either oral or parenteral administration,but parenteral administration is preferred. Specific examples compriseinjections, suppositories, nasal administration, intrapulmonaryadministration, and percutaneous administration. Injections may beadministered locally or systemically by, for example, intravenousadministration, intramuscular administration, intraperitonealadministration, subcutaneous administration, intravenous drip infusion,or such. When aiming for administration by injection, the dosage form ofthe compositions of the present invention can take forms of appropriatecombinations with sterile water or physiological saline, emulsifiers,suspending agents, detergents, stabilizers, vehicles, preservatives, andsuch to formulate effective amounts of the antibodies of the presentinvention. When aiming for administration as oral preparations,capsules, granules, suspensions, powders, tablets, emulsions, solutions,and such can be selected as the dosage form of the compositions of thepresent invention.

Examples of emulsifiers or detergents described above comprise stearyltriethanolamine, sodium lauryl sulfate, lauryl amino propionate,lecithin, glyceryl monostearate, sucrose fatty acid ester, and glycerinfatty acid ester.

Examples of suspensions described above comprise, in addition to thedetergents described above, for example, hydrophilic polymers such aspolyvinyl alcohol, polyvinylpyrrolidone, methylcellulose,hydroxymethylcellulose, hydroxyethylcellulose, andhydroxypropylcellulose.

Examples of stabilizers described above comprise those generally used inmedicine.

Examples of vehicles described above comprise those generally used inmedicine such as liposomes, microspheres, and lipid vesicles.

Examples of preservatives described above comprise methylparaben,propylparaben, chlorobutanol, benzyl alcohol, phenethyl alcohol,dehydroacetic acid, and sorbic acid.

Sterile compositions for injection can be formulated according to usualformulation operations using oils for injection or aqueous solutions forinjection, such as distilled water for injection. Examples of aqueoussolutions for injection comprise physiological saline, isotonicsolutions comprising glucose or other adjuvants, for example,D-sorbitol, D-mannose, D-mannitol, and sodium chloride. These may beused in combination with appropriate solubilizers, for example,alcohols, specifically ethanol; polyalcohols, for example propyleneglycols and polyethylene glycols; nonionic detergents, for examplepolysorbate 80™ and HCO-50.

Examples of oils for injection comprise sesame oils and soybean oils,which may be used in combination with solubilizers, such as benzylalcohol or benzyl benzoate. They may also be combined with buffers, forexample, phosphate buffers and sodium acetate buffers; soothing agents,for example, procaine hydrochloride; stabilizers, for example, benzylalcohol and phenol; and antioxidants.

Prepared injections are usually loaded into appropriate ampules.Alternatively, the injections may be prepared in forms where adequateamounts of appropriate vehicles, such as aqueous solutions and oils forinjection, are added to ampules containing the freeze-dried antibodiesof the present invention at the time of use.

The dose of inhibitors of the present invention just needs to be anamount sufficient to suppress the adhesion of Th1 cells in pathologicallesions developed by the effect of Th1 cells, and the doses varydepending on the patient's age, sex, weight, symptoms, and such, as wellas the therapeutic purpose, the administration method, and the like. Oneskilled in the art can determine appropriate doses in consideration ofthese factors and the activity of selected antibodies. For example, thedoses can be selected from a range of one to five times per day, andfrom a range of 0.0001 to 1000 mg/kg weight, preferably 0.01 to 100mg/kg weight, and more preferably 0.1 to 10 mg/kg weight peradministration. Alternatively, the dose can be selected, for example,from a range of 0.001 to 100000 mg/body, preferably 0.1 to 10000mg/body, more preferably 1 to 100 mg/body per patient.

All publications cited herein are incorporated by reference into thisdescription.

EXAMPLES

Hereinbelow, the present invention will be specifically described withreference to Examples, but it is not to be construed as being limitedthereto.

Example 1 Production of Cells Expressing the Full-Length AdhesionMolecules

The full length cDNAs of the adhesion molecules JAM-A, JAM-B, JAM-C,ESAM, and CAR were cloned as described below. A mouse heart cDNA library(Clontech Quick Clone 7133-1) was used as template for JAM-A, JAM-B, andJAM-C; mouse small intestine cDNAs were used as template for ESAM; andmouse spleen cDNAs were used as template for CAR. The primers for eachwere designed based on GenBank™ sequences (JAM-A (U89915), JAM-B(AF255911), JAM-C (AJ300304), ESAM (AF361882), and CAR (NM009988)) foramplification by PCR. For example, the primers shown below were used forCAR:

mCAR F1: (SEQ ID NO: 5) GCGGTCGACGCCACCATGGCGCGCCTACTGTGCTTCGTGCT mCARR2: (SEQ ID NO: 6) CGCCGCGGCCGCTTATACCACTGTAATGCCATCGGTCT

The obtained cDNA fragments were inserted into the expression vectorpMXII IRES-EGFP (Oncogene (2000) 19(27):3050-3058) and introduced intothe 293/EBNA-1 cell line (Invitrogen) together with the packaging vectorpCL-Eco (Imgenex, San Diego, Calif.) to produce recombinantretroviruses. The B300 cell line was infected with this viral solution,and expressing cells were obtained after separating EGFP-positive cellsby cell sorting. The 293/EBNA-1 cell line was cultured in Dulbecco'smodified Eagle's medium containing 10% FCS, while the B300 cell line wascultured in RPMI-1640 containing 10% FCS and 55 μM 2-mercaptoethanol.

Example 2 Preparation of Activated Lymphocytes

Mouse spleens were ground on a 100 μm mesh and erythrocytes werehemolyzed to obtain splenocytes. CD4-positive cells were isolated fromthe splenocytes using MACS (Miltenyi Biotec). 4×107 splenocytes weresuspended in 70 μl of PBS containing 1% FCS and 2 mM EDTA, and treatedwith FcR blocking reagent (Miltenyi Biotec). Anti-CD4antibody-immobilized magnetic beads were added in and the reaction wascarried out at 4° C. for 20 minutes. After washing, positive selectionwas carried out using AutoMACS. The proportion of CD4-positive cells was90% or more. The purified CD4-positive cells were added to plates withanti-CD3 antibody immobilized at 1 μg/ml, and cultured in the presenceof 10 μg/ml anti-CD28 antibody. After two days, 20 ng/ml IL-2 was addedand cultured, and activated lymphocytes at seven to nine days afteractivation were used in experiments.

Example 3 Production of Chimeric Proteins Between Alkaline Phosphataseand the Extracellular Region of Adhesion Molecules

First, the pcDNA3.1(+)-SEAP(His)10-Neo vector was produced as follows:the intrinsic SalI site of pCDNA3.1 (+)-Neo vector (CLONTECH) wasdeleted by SalI digestion followed by blunting. The cDNA fragment SEAP(His)10 was amplified by PCR using pDREF-SEAP His6-Hyg (J. Biol. Chem.,1996, 271, 21514-21521) as template and primers to which HindIII andXhoI have been attached to the 5′ end and 3′ end, respectively. Theresulting cDNA fragments were digested with HindIII and XhoI, theninserted into the pCDNA3.1 (+)-Neo vector from which the SalI site hasbeen deleted.

Next, the extracellular regions of the adhesion molecules were amplifiedby PCR using the full-length cDNAs obtained in Example 1 as templatesand primers to which SalI and NotI have been attached to the 5′ end and3′ end, respectively (for example, the primers below were used for CAR).

mCAR F1: (SEQ ID NO: 5) GCGGTCGACGCCACCATGGCGCGCCTACTGTGCTTCGTGCT mCARR1: (SEQ ID NO: 7) CGCCGCGGCCGCTCGGTTGGAGGGTGGGACAACGTCTA

The amplified cDNA fragments were digested with SalI and NotI, theninserted into the pcDNA3.1(+)-SEAP(His)10-Neo vector described above.The constructs enable the expression of secretory chimeric proteins(hereinafter referred to as AP chimeric proteins) in which theextracellular domains of the adhesion molecules are linked with humansecretory placental alkaline phosphatase via a three-amino acid linker(Ala-Ala-Ala), and having a tag of ten histidines (His)10 at theC-terminal. The obtained expression vectors for the AP chimeric proteinswere introduced into the 293/EBNA-1 cell line using TransIT LT1 (TAKARA#V2300). The cells were cultured for four to five days. The AP chimericproteins secreted into the culture supernatant were collected bycollecting the culture supernatants by centrifugation and filteringthrough a 0.22 μm filter. Hepes (pH 7.4) and sodium azide were addedthereto until a final concentration of 20 mM and 0.02%, respectively,and the supernatants were stored at 4° C. The concentrations of APchimeric proteins were calculated after measuring the alkalinephosphatase activity using the Great EscApe Detection kit (CLONTECH#K2041-1).

Example 4 Adhesion of Activated Lymphocytes to Immobilized CAR

Cell adhesion experiments were carried out using AP chimeric proteins toidentify molecules functioning as adhesion molecules that adhere tolymphocytes. First, 50 μl of 10 μg/ml anti-alkaline phosphatase antibody(Seradyn MIA1802) was added to 96 well ELISA plates (Nunc), and allowedto stand at 37° C. for 30 minutes to immobilize the antibody. Afterwashing the plates with PBS, the non-specifically binding sites wereblocked with BlockAce (Dainippon Pharmaceutical Co.). AP chimericproteins were diluted to a final concentration of 10 nM, added to thewells, and allowed to stand at room temperature for 30 minutes toimmobilize the proteins. Activated lymphocytes were suspended in celladhesion buffer (RPMI 1640, 0.5% BSA, and 20 mM HEPES (pH 7.4)),fluorescently labeled with Calcein-AM (Dojin), then added to wells at1×10⁵ cells/well, and incubated at 37° C. for one hour. The non-adherentcells were washed away. A cell lysis solution (10 mM TrisHCl (pH 8.0),1% TritonX-100) was added, then adherent cells were quantified usingWallac ARVO SX 1420 MULTILABEL COUNTER (Perkin Elmer) and measuring atan excitation wavelength of 485 nm and detection wavelength of 535 nm.The degree of cell adhesion was represented as a percentage ratio ofadherent cells to added cells. The extracellular AP chimeric proteinsderived from JAM-A, JAM-B, JAM-C, CAR, and ESAM were immobilized, and asa result, CAR was revealed to function as an adhesion molecule thatadheres to activated lymphocytes (FIG. 1).

Example 5 Production of Monoclonal Antibodies Against CAR

CAR-AP chimeric proteins were purified for use as antigens forimmunization. Purification was carried out using the histidine tags onthe C-terminus of the AP chimeric proteins with His Trap Kit (AmershamBiosciences #17-1880-01). Culture supernatant containing a CAR-APchimeric protein was loaded onto 1 ml HiTrap Chelating HP Column(Amersham #17-0408-01). After washing with a 20 mM imidazole solution,CAR-AP chimeric proteins were eluted from the column using a 500 mMimidazole solution. The concentration of CAR-AP chimeric proteins wascalculated from the enzyme activity measurement using the Great EscApeDetection kit (CLONTECH #K2041-1) and protein quantitation using theProtein Assay kit II (BIO-RAD 500-0002JA).

The obtained CAR-AP chimeric protein was combined with TiterMax, andthen used to immunize rats. Lymphocytes were isolated from the immunizedrats, mixed such that the ratio of P3 myeloma cells to lymphocytes wouldbe 1:5, and cells were fused using a PEG1500 solution (783-641;Boehringer). Hybridomas were selected using HAT medium (GIBCO BRL31062-011) and culture supernatants of the obtained hybridomas werescreened by sandwich ELISA using CAR-Fc chimeric proteins. Cells frompositive wells were cloned, and three types of clone (4C9, 5E9, and5G11) were obtained. Specificity was examined by FACS and as a result,clone 5G11 reacted only with B300 cells expressing CAR, and did notreact with cells of the B300 parental line nor to B300 cells expressingJAM-A, JAM-B, JAM-C, or ESAM (FIG. 2A). The same results were obtainedfor clones 4C9 and 5E9.

Example 6 Cell Adhesion-Inhibiting Activity of Anti-CAR Antibodies

To investigate the effect of antibodies on cell adhesion mediated byhomophilic binding of CAR, 10 μg/ml anti-CAR antibodies were added toB300/CAR cells and the immobilized CAR-AP chimeric proteins, pretreatedat room temperature for ten minutes, then the cell adhesion activity inthe presence of antibodies was examined. As a result, the adhesion ofB300/CAR cells to CAR-AP chimeric proteins was suppressed by antibody5G11 and increased by antibodies 4C9 and 5E9. The adhesion of activatedlymphocytes to CAR-AP chimeric proteins was suppressed by antibodies 4C9and 5G11, while an increase by antibody 5E9 was not observed (FIGS. 2Band 2C).

Example 7 Existence of Unknown CAR Ligands in Activated Lymphocytes

The present inventors examined whether activated lymphocytes expressedCAR and adhered to immobilized CAR-AP chimeric proteins via a homophilicbinding. First, the expression of CAR on cell surfaces was examinedusing anti-CAR antibodies and FACS. Activated lymphocytes were reactedwith antibody 5G11, then reacted with PE-labeled goat anti-rat IgGantibodies and assayed using FACSCalibur (Becton Dickinson). However,CAR expression was not detected on the surface of activated lymphocytes(FIG. 3A). Next, the expression of CAR mRNA was examined using RT-PCR;however, mRNA expression was also not detected (FIG. 3B). Therefore, CARis not expressed in activated lymphocytes, and the existence of anunknown CAR ligand(s) on the surface of lymphocytes was suggested.

Given this, first the possibility that the unknown CAR ligand was anintegrin was examined. The present inventors examined the effects ofantibodies against CD11a (M17/4 eBioScience 14-0111), CD18 (GAME-46 BD555280), CD29 (2C9.G2 BD 553343), CD49d (R1-2 BD 553153), CD51 (RMV-7 BD552299), and CD61 (GAME-46 BD 555280) on cell adhesion. However, none ofthe antibodies inhibited adhesion between CAR and activated lymphocytes.

Example 8 Cell Adhesion Activity of Various Lymphocytes to ImmobilizedCAR-AP Chimeric Proteins, and Binding Activity of Various Lymphocytes toCAR-AP Chimeric Proteins

Next, the characteristics of lymphocytes that adhere to CAR wereinvestigated. The adhesion activity to CAR-AP chimeric proteins ofCD4-positive cells (resting CD4+ cells) immediately after purificationwith MACS, and of activated lymphocytes further cultured for five daysin the presence of 20 ng/ml IL-2 or 25 ng/ml IL-15 two days afteractivation with anti-CD3 antibody, was examined. As a result, theactivated lymphocytes which were stimulated with IL-2 adhered, butneither the activated lymphocytes stimulated with IL-15, nor the restingCD4+ cells adhered. The TK1 cell line also did not adhere (FIG. 4).

Next, the binding activity of CAR-AP chimeric proteins to the cellsurface of various lymphocytes was investigated. The cells were reactedwith 40 nM CAR-AP chimeric proteins at 4° C. for 30 minutes, thenreacted with FMAT Blue-labeled mouse anti-alkaline phosphatase antibodyat 4° C. for 30 minutes. Binding was detected by FACS. The resultsrevealed that CAR-AP chimeric proteins bound to activated lymphocyteswhich were stimulated with IL-2, but did not bind to activatedlymphocytes which were stimulated with IL-15, nor to resting CD4+ cellsnor to the TK1 cell line (FIG. 5).

Example 9 Identification of the Novel Ligand CARL (CAR Ligand) for CAR

Next, the present inventors investigated on the possibility that theunknown ligand for CAR is an IgSF. First, the positional relationshipsof CAR and JAM family molecules on the mouse chromosomes wereinvestigated. A mouse Ensembl database was searched, and as a result,these molecules existed as discrete clusters formed on chromosomes 1, 9and 16. Given this, 116 IgSFs existing near CAR and JAM family moleculeswere focused on and their expression in various lymphocytes wasinvestigated by real-time PCR. Using an RNeasy mini kit (Qiagen, Hilden,Germany), total RNAs were isolated from 5×10⁶ TK1 cells, IL-2-stimulatedactivated lymphocytes, and IL-15-stimulated activated lymphocytes. Thereal-time PCR was carried out in the presence of SYBR-green using ABI7700 Sequence Detection System (PE Applied-Biosystems). As a result,ENSMUSG0000048534 showed an expression pattern correlating to theactivity of adhesion to CAR. Cells expressing this molecule wereproduced to examine whether ENSMUSG0000048534 was an unknown CAR ligand.The expressing cells were obtained by the method described in Example 1using as a template the cDNAs of IL-2-stimulated activated lymphocytesand primers were designed as shown below based on the sequence ofENSMUSG0000048534:

mCARL F1: (SEQ ID NO: 8) GCGGTCGACGCCACCATGCTTTGCCTCCTGAAACTGATTGTGmCARL R2: (SEQ ID NO: 9) CGCGGCGGCCGCTTACTTGGATCTGACTGAAGAGCGGAC

Chimeric proteins between AP and the extracellular regions of adhesionmolecules (JAM-A, JAM-B, JAM-C, CAR, and ESAM) were prepared similarlyas in Example 3. To investigate the homophilic binding of the ligandencoded by ENSMUSG0000048534, a chimeric protein of this ligand with APwas similarly prepared using the following primers:

mCARL F1: (SEQ ID NO: 8) GCGGTCGACGCCACCATGCTTTGCCTCCTGAAACTGATTGTGmCARL R1: (SEQ ID NO: 10) [ID:1830]CGCGGCGGCCGCATTTCCATTCAGGATGCCCTGCTGACC

When the adhesion activities to the extracellular AP chimeric proteinsof various adhesion molecules were examined using these expressingcells, the cells only adhered to CAR (FIG. 6A). Furthermore, when theactivities of CAR-expressing B300 cells in adhering to the AP chimericproteins of these extracellular regions were examined, theCAR-expressing B300 cells adhered, and the adhesion was inhibited by 4C9and 5G11 (FIG. 6B). The results described above revealed thatENSMUSG0000048534 was a novel CAR ligand on lymphocytes and the ligandwas named CARL (CAR ligand).

Example 10 Properties of CARL

CARL is a molecule comprising 379 amino acids and belonging to IgSF, andhas two extracellular Ig-like domains (FIG. 7). The gene for CARL islocated between genes for epithelial V-like antigen 1(ENSMUSG0000032092) and sodium channel beta-2 subunit(ENSMUSG0000037714) on mouse chromosome 9. CARL matched with BCO50133(gi29477100), which is deposited as “similar to AMICA”. When the NCBI nrwas searched for the nucleotide sequence of CARL using blastn, highhomology to XM_194453 (gi38089807) in addition to BCO50133 (gi29477100)was shown. XM_194453 (gi38089807) is a mutant of BCO50133 (gi29477100),in which the first eleven amino acids are replaced with 37 amino acids,and an arginine at position 231 is replaced with glutamine. Moreover,according to the orthologue prediction in the mouse Ensembl,ENSG00000160593 deposited as “AMICA” was predicted to be a humanorthologue of CARL. There are two types of ENSG00000160593 transcripts,and they (ENST00000292067 and ENST00000303475) are 5′-end splicingvariants. A comparison between their deduced amino acid sequences showedthat instead of the first 14 amino acids of ENST00000292067, fourdifferent amino acids were inserted in ENST00000303475. Homology betweenCARL and AMICA (ENST00000292067 and ENST00000303475) was 38.2% and 37.7%at the amino acid level, respectively. Since the human AMICA locus islocated on chromosome 11 (11q23.3), a region homologous to mouse CARL onthe human chromosome, human AMICA is thought to be human CARL, a humanhomologue for mouse CARL. Recently, human JAML (AJ515553) was reportedas an adhesion molecule expressed in bone marrow-derived cells (Blood,2003 (102), 3371-3378), but this molecule is a mutant lacking the 37nucleotides from 560 to 596 bp of human CARL (ENST00000292067) andhaving this sequence inserted between 693 and 694 bp. Like human CARL,human JAML comprises of 394 amino acids, but the amino acid sequence ofhuman JAML at position 94, and position 161 to 205 are completelydifferent from human CARL.

Example 11 Protein-Protein Interaction between CAR and the ExtracellularRegion of CARL, and their Dissociation Equilibrium Constant

To examine the protein-protein interaction and affinity between CAR andthe extracellular domain of CARL, the value of the dissociationequilibrium constant Kd was determined with a biosensor that uses thesurface plasmon resonance phenomenon using BIAcore X (BIAcore), with thefollowing procedure: first, anti-alkaline phosphatase antibodies(Seradyn) at 50 μg/ml were immobilized onto sensor chip CM5 (BIAcore)using the amino-coupling method. Then, a culture supernatant comprisingCAR-AP chimeric protein or control AP chimeric protein diluted to 20 nMwith HBS-EP buffer (BIAcore) was added to immobilize 500 RU of APchimeric proteins onto the sensor chip. The protein-protein interactionswere measured by adding culture supernatants comprising CARL-Fc chimericproteins diluted to 20 nM, 6 nM, or 0.6 nM with HBS-EP buffer (BIAcore)at 20 μl/min for 210 seconds. From the binding rate constant anddissociation rate constant obtained using BIAevaluation software version3.2 (BIAcore), the dissociation equilibrium constant between CAR andCARL was revealed to be 4.8 nM (FIG. 8).

Example 12 Selective Expression of CARL in Th1 Cells and SelectiveAdhesion of Th1 Cells to CAR

Th1 and Th2 cells were prepared to investigate the expression of CARL inTh1 and Th2 cells. CD4-positive cells purified with MACS were added toplates onto which anti-CD3 antibodies at 1 μg/ml have been immobilized,and were then stimulated in the presence of 10 μg/ml anti-CD28antibodies. The cells stimulated with anti-CD3 antibodies in thepresence of 10 ng/ml IL-12 and 10 μg/ml anti-IL-4 antibodies (MP4-25D2;PharMingen) in the case of differentiation into Th1 cells, and in thepresence of 15 ng/ml IL-4 and 15 μg/ml anti-IL-12 antibodies (24910.1; R& D Systems) in the case of differentiation into Th2 cells. After twodays, the cells differentiated into Th1 were cultured in the presence of20 ng/ml IL-2 and 10 ng/ml IL-12, while the cells differentiated intoTh2 were cultured in the presence of 20 ng/ml IL-2 and 15 ng/ml IL-4.The cells were used in experiments seven to nine days after thestimulation. Cell differentiation into Th1 and Th2 was confirmed basedon IFN-γ and IL-4 production, respectively. The expression of CARL andHPRT (control) mRNAs was investigated by real-time PCR using RNAobtained from these cells. The results revealed that CARL wasselectively expressed in Th1 cells (FIG. 9A). When the cell surfacebinding activity of CAR-AP chimeric proteins was examined, a selectivebinding to Th1 cells was similarly observed (FIG. 9B). Furthermore, whenthe adhesion activity of Th1 and Th2 cells to CAR-AP chimeric proteinswas examined, it was revealed that only Th1 cells selectively adhered toCAR, matching with the expression of CARL (FIG. 9C). Adhesion wasinhibited by 4C9 and 5G11 (FIG. 10A).

Further, CARL expression was analyzed in cells other than CD4-positive Tcells. Cells were prepared from the spleens and peripheral blood ofC57BL/6 male mice and suspended in FACS buffer (PBS/1% FBS/1 mM EDTA)containing 5% mouse serum and 5% rat serum. An FcR blocking solution wasadded thereto, and incubation was carried out on ice for ten minutes.Then, fluorescently labeled anti-mCARL antibody was reacted on ice for30 minutes with fluorescently labeled antibodies against various cellline markers, and measurements were carried out using FACScalibur.

As a result, in the immune tissues of normal mice, CARL expression wasdetected in CD3-positive T cells, CD11b-positive myeloid cells,CD11c-positive dendritic cells, and B220-positive B cells prepared fromthe spleen, but not in DX5-positive NK cells and F4/80-positivemonocytes/macrophages. Furthermore, the expression of CARL wasdetectable in SSC high/Gr1-positive neutrophils prepared from peripheralblood, but not in SSC high/F4/80-positive eosinophils (FIG. 10B).

Example 13 Preparation of Cells Expressing CARL Lacking Ig-Like Domains

Cells expressing mCARL lacking Ig-like domains were produced todetermine the mCAR-binding region in CARL. Expression vectors weremodified as described below. First, the modified vector pMX MCS2.2 IRESPuro was constructed by newly inserting a multi-cloning site(cgcggatcctaattaattaaggataaactgtcgacgaattcgcggccgccacgcgttcgcga; SEQ IDNO:19) and IRES/Puro between the BamHI and SalI of pMX (J. Biol. Chem.,2002, 277, 5583-5587), using the same method as described in Oncogene(2000) 19(27):3050-3058. Then, the modified vector pMX ssFLAG MCS2.2IRES Puro was produced by inserting a signal sequence and FLAG tagbetween BamHI and SalI of pMX MCS2.2 IRES Puro.

The signal sequence of CARL was presumed to be the amino acids fromposition 1 to 20; the first Ig-like domain (domain 1) was presumed to bethe amino acids from position 30 to 139; and the second Ig-like domain(domain 2) was presumed to be the amino acids from position 143 to 254(SignalP, on the world wide web at cbs.dtu.dk/services/SignalP/; SMART,on the world wide web at smart.embl-heidelberg.de/). Thus, thefull-length CARL indicates a protein comprising the amino acids fromposition 21 to 379; domain 1-lacking CARL indicates a protein comprisingthe amino acids from position 21 to 29 and from position 140 to 379; anddomain 2-lacking CARL indicates a protein comprising the amino acidsfrom position 21 to 139 and from position 255 to 379.

The full-length CARL and domain 1-lacking CARL were cloned by PCRamplification using the full-length CARL cDNA obtained in Example 9 as atemplate, and then inserting the obtained cDNA fragments into theexpression vector pMX ssFLAG MCS2.2 IRES Puro. The domain 2-lacking CARLwas cloned by amplifying a cDNA fragment by two-step PCR using primerscomprising the nucleotide sequences corresponding to the amino acidsequence from position 134 to 139 and to the amino acid sequence fromposition 255 to 260 as well as the complementary sequences. The B300cell line was infected with a recombinant retrovirus produced by themethod described in Example 1 and expressing cells were obtained byselecting the gene-introduced cells with 5 μg/ml puromycin. Theexpression of CARLs lacking the Ig-like domains on the surface of cellswas confirmed using anti-FLAG antibody (SIGMA) (FIG. 11A). The adhesionactivity of the cells to CAR-AP chimeric proteins was examined by themethod described in Example 4. B300 cells expressing full-length CARLand B300 cells expressing CARL lacking domain 2 adhered to CAR-APchimeric proteins, while B300 cells expressing CARL lacking domain 1 didnot (FIG. 11B). Thus, domain 1 of CARL was revealed to be essential forthe binding between CAR and CARL.

Example 14 Preparation of Monoclonal Antibodies Against CARL, andExpression in Th1 Cells and Cell Adhesion Inhibition Activity UsingAnti-CARL Antibodies

Anti-CARL antibody #3 was obtained by the method described in Example 5,using as an immunization antigen the CARL-AP chimeric protein obtainedin Example 9. Specificity was examined by FACS and the results showedthat #3 antibody reacted with CARL-expressing B300 cells but not withthe parental cell line B300 (FIG. 12A). When CARL expression in Th1 andTh2 cells prepared by the method described in Example 12 was examinedusing #3 antibody, CARL was strongly and selectively expressed in Th1cells (FIG. 12B). Meanwhile, #3 antibody inhibited the adhesion ofCAR-expressing B300 cells to CARL-AP proteins (FIG. 12C). Whenimmunostaining was carried out using the cells obtained in Example 13,#3 antibody bound to CARL on the cell membrane of B300 cells expressingthe full-length CARL and B300 cells expressing CARL lacking domain 2,but not to the CARL of B300 cells expressing CARL lacking domain 1. Thus#3 antibody, which has the activity of inhibiting binding, was revealedto recognize domain 1, which is required for the binding between CAR andCARL.

Example 15 Binding Between Human CAR and Human CARL

Cells expressing a full-length human CAR and cells expressing afull-length human CARL, as well as AP chimeric proteins with theextracellular region of human CAR and AP chimeric proteins with theextracellular region of human CARL were generated to verify that thephenomena observed in mice is similarly observed in humans. Theexpression vector used was pMX MCS2.2 IRES Puro, obtained in Example 13.

The full-length cDNAs for human CAR and CARL were cloned as follows:cDNAs synthesized from human whole brain (Clontech) were used astemplate. The primers used were designed as shown below, based on thesequences in GenBank™ (human CAR (NM_001338) and human CARL (AY138965)),and amplification was carried out by PCR.

hCAR F1: (SEQ ID NO: 11) CGCGTCGACATGGCGCTCCTGCTGTGCTTCGTG hCAR R2: (SEQID NO: 12) GCGGGCGGCCGCCTATACTATAGACCCATCCTTGCT hCARL F1: (SEQ ID NO:13) CGCGTCGACATGTTTTGCCCACTGAAACTCATC hCARL R2: (SEQ ID NO: 14)GCGGGCGGCCGCTCAAAAGGCTTGCTGTGTTTTTGG

Each of the obtained cDNA fragments was inserted into the expressionvector pMX MCS2.2 IRES Puro, and recombinant retroviruses were producedby the method described in Example 1. Expressing cells were obtained byselecting the gene-introduced B300 cell lines with 5 μg/ml puromycin. APchimeric proteins with the extracellular domains of human CAR and CARLwere generated by the method described in Example 3, using the followingprimers:

hCAR F1: (SEQ ID NO: 11) CGCGTCGACATGGCGCTCCTGCTGTGCTTCGTG hCAR R1: (SEQID NO: 15) GCGGGCGGCCGCTTTATTTGAAGGAGGGACAACGTT hCARL F1: (SEQ ID NO:13) CGCGTCGACATGTTTTGCCCACTGAAACTCATC hCARL R1: (SEQ ID NO: 16)GCGGGCGGCCGCATTACCACCCAAGACCAGAGGCCT

The expression of human CAR on the surface of human CAR-expressing B300cells was confirmed using an anti-human CAR antibody (UpstateBiotechnology), while the expression of human CARL on the surface ofhuman CARL-expressing B300 cells was confirmed using human CAR-APchimeric proteins (FIG. 13A). When, the adhesion activity of humanCAR-expressing B300 cells to human CARL-AP chimeric proteins wasexamined by the method described in Example 4, binding between human CARand human CARL was detected. Likewise, when the adhesion activity ofhuman CARL-expressing B300 cells to human CAR-AP chimeric proteins wasexamined, binding between human CAR bound to human CARL was detected(FIG. 13B).

Example 16 Generation of Monoclonal Antibodies Against Human CARL andCell Adhesion-Inhibiting Activity of Anti-Human CARL Antibodies

Four types of mouse anti-human CARL antibodies (#5, #49, #77, and #85)were obtained by the method described in Example 5, using asimmunization antigens the human CARL-AP chimeric proteins obtained inExample 15. Specificity was examined by FACS, and as a result, theculture supernatant of #5 reacted with B300 cells expressing human CARL,but not with the parental cell line B300 (FIG. 14A). Similar resultswere obtained with clones #49, #77, and #85. Of these antibodies, theculture supernatants of #5, #49, and #77 inhibited the adhesion of humanCAR-expressing B300 cells to human CARL-AP chimeric proteins (FIG. 14B).

Example 17 Therapeutic Effect of Anti-CARL Antibody on ContactDermatitis

Contact hypersensitivity (CHS) was induced in 6-week-old mice asfollows: first, mice were sensitized five and four days before CHSinduction by applying 25 μl of 0.4% dinitrofluorobenzene (WAKO) onto theabdomen of each mouse. Anti-CARL antibody #3 was administered at a doseof 500 μg per mouse from the caudal vein five days before and on the dayof CHS induction. Then, CHS was induced by applying 20 μl of 0.4%dinitrofluorobenzene per mouse onto the ear auricles. The thickness ofright and left auricles was measured 24 hours after induction. Theswelling of auricles was suppressed by the antibody treatment (FIG. 15).

As shown in Example 12, CARL expression was observed in activated Th1cells and neutrophils, and the anti-CARL antibody was thought tosuppress CHS by suppressing the interaction between CARL and CAR.

INDUSTRIAL APPLICABILITY

The present invention provides methods for detecting Th1 cells. Th1cells are CD4+ T lymphocytes, and when activated they secrete cytokinessuch as IL-2, IL-12, tumor necrosis factor (TNF) β, lymphotoxin (LT),and IFN-γ. Th1 cells are chiefly involved in cellular immunity. Th1 isthought to play a major role in the onset of organ-specific autoimmunediseases, comprising rheumatoid arthritis, Crohn's disease, type Idiabetes, and multiple sclerosis. Moreover, selective imbalances orimbalanced activation of Th1 or Th2 cells is thought to be a cause ofsome chronic inflammatory or allergic diseases. On the other hand, theTh2 response dominates in diseases such as cancer, allergies (atopicdisease and such), and parasite infections; however, it has beensuggested that these diseases may be eased by inducing the Th1 response.Specifically, it has been reported that a shift to the Th1 phenotypeincreases the secretion of IFNα, IFN β and IFN γ, and IL-12, IL-18, andsuch, and strengthens host immunological defense against intracellularpathogens, such as viruses. Given this, the methods of the presentinvention for detecting Th1 cells can be used, for example, to diagnoseor elucidate diseases in which a selective imbalance or imbalancedactivation of Th1 or Th2 cells, or such, is involved. The methods canalso be used to assess therapeutic effects on such diseases.

Furthermore, the present invention relates to methods of screening forinhibitors of the binding between CAR and CARL. Inhibitors of thebinding between CAR and CARL comprise antibodies and inhibit the celladhesion between Th1 cells expressing CARL and epithelial cells andendothelial cells expressing CAR. These inhibitors and compositionscomprising these inhibitors are thus expected to suppress the adhesionof Th1 cells. With these inhibitors and compositions comprising theinhibitors, therapeutic effects on autoimmune diseases in which Th1cells are involved are expected.

The present invention also provides antibodies that inhibit the bindingbetween CAR and CARL. Like the substances described above, suchantibodies are expected to suppress the adhesion of Th1 cells, tosuppress cell adhesion in, for example, contact dermatitis, and toalleviate diseases. Moreover, since the binding between CAR and CARL isthought to be involved in the infiltration of Th1 cells, they areexpected to be useful for elucidating the mechanism of cellinfiltration.

The invention claimed is:
 1. A method of treating contact dermatitis ina human subject in need thereof, the method comprising administering tothe human subject in need thereof a therapeutically effective amount ofan antibody that specifically binds to a protein consisting of the aminoacid sequence of SEQ ID NO:2, wherein the antibody binds to domain 1(amino acids 27-136 of SEQ ID NO:2) and inhibits binding between theprotein consisting of the amino acid sequence of SEQ ID NO:2 and aprotein consisting of the amino acid sequence of SEQ ID NO:17.
 2. Themethod of claim 1, wherein the antibody is a human antibody.
 3. Themethod of claim 1, wherein the antibody is a humanized antibody.
 4. Themethod of claim 1, wherein the antibody is a chimeric antibody.
 5. Themethod of claim 1, wherein the antibody is a single chain antibody. 6.The method of claim 1, wherein the antibody is an antibody fragmentselected from the group consisting of an Fab fragment, an Fab′ fragment,an F(ab′)₂ fragment, and an Fv fragment.